Nucleic acid amplification using microfluidic devices

ABSTRACT

The present invention provides microfluidic devices and methods using the same in various types of thermal cycling reactions. Certaom devices include a rotary microfluidic channel and a plurality of temperature regions at different locations along the rotary microfluidic channel at which temperature is regulated. Solution can be repeatedly passed through the temperature regions such that the solution is exposed to different temperatures. Other microfluidic devices include an array of reaction chambers formed by intersecting vertical and horizontal flow channels, with the ability to regulate temperature at the reaction chambers. The microfluidic devices can be used to conduct a number of different analyses, including various primer extension reactions and nucleic acid amplification reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/281,960, filed Apr. 6, 2001, U.S. Provisional Application No.60/300,516, filed Jun. 22, 2001, and U.S. Provisional Application No.60/334,473, filed Nov. 16, 2001, each of which is incorporated herein byreference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with support from Grant Number CTS-0088649awarded by the National Science Foundation. Therefore, the U.S.government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to microfluidic devices and methods ofusing the same in various types of thermal cycling reactions.

BACKGROUND OF THE INVENTION

Nucleic acid amplification reactions have emerged as powerful tools in avariety of genetic analyses and diagnostic applications. The value ofthese techniques is their ability to rapidly increase the concentrationof target nucleic acids of interest that might be present at very lowand otherwise undetectable levels. For instance, by utilizing thepolymerase chain reaction (PCR) amplification technique, one can amplifya single molecule of a target nucleic acid by 10⁶ to 10⁹.

PCR is perhaps the most well-known of a number of differentamplification techniques. This well established procedure involves therepetition of heating (denaturation) and cooling (annealing) cycles inthe presence of a target nucleic acid, primers that hybridize to thetarget, deoxynucleotides, a polymerase and cofactors such as metal ions.Each cycle produces a doubling of the amount of the target DNA. Thecycles are conducted at characteristic temperatures: 95° C. fordenaturing double stranded nucleic acid, 50 to 65° C. for hybridizationof primer to the target nucleic acid, and 72 to 77° C. for primerextension (see, generally, Sambrook et al., (1989) Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; seealso U.S. Pat. Nos. 4,683,202 and 4,683,195, for example).

Methods for conducting PCR amplifications fall into two general classes.The approach typically utilized is a time domain approach in which theamplification reaction mixture is kept stationary and the temperature iscycled (see, e.g., Cheng, et al. (1996) Nucleic Acids Res. 24:380-385;Shoffer, et al. (1996) Nucleic Acids Res. 24:375-379; and Hong, et al.(2001) Electrophoresis 22:328-333). While methods utilizing thisapproach can be conducted with relatively small sample volumes, themethods require complex regulation of heater elements and relativelylong reaction times. Another approach that has been discussed is limitedto a space domain approach in which three temperature zones areconstantly kept at the different temperatures and the reaction mixtureruns in a serpentine flow channel above it (see, e.g., Kopp et al.(1998) Science 280:1046-1048). A method such as this can be conducted atrelatively high speed because it is not necessary to heat and cool theheaters, but requires the use of relatively large sample volumes.

SUMMARY OF THE INVENTION

A variety of microfluidic devices and methods for conducting temperaturecontrolled analyses are provided herein. Unlike conventionalmicrofluidic devices, the devices disclosed herein include elastomericcomponents. In some instances, much of the device is manufactured fromelastomeric materials. The devices can be utilized in a wide variety ofapplications, particularly in analyses involving thermal cycling.

Certain of these microfluidic devices include (a) a substrate comprisingan elastomeric material; (b) a flow channel disposed within thesubstrate, the flow channel being configured such that a sampleintroduced into the flow channel can be cycled around the flow channel,and comprising a plurality of temperature regions at which temperaturecan be regulated, each temperature region located at a differentlocation along the flow channel; (c) an inlet in fluid communicationwith the flow channel via which the sample can be introduced into theflow channel; and (d) a temperature controller operatively disposed toregulate temperature within at least one of the plurality of temperatureregions. Devices of this type can also include one or more pumps fortransporting fluid through the flow channel. Certain of these pumpscomprise one or more control channels, each of the control channels ofthe pump formed within an elastomeric material and separated from theflow channel by a section of an elastomeric membrane, the membrane beingdeflectable into or retractable from the substantially circular flowchannel in response to an actuation force applied to the controlchannel. The flow channel in some of these devices is substantiallycircular. A flow channel of this shape cannot be formed withconventional silicon-based microfluidic devices that utilize electricalcurrent to move solutions through the microfluidic channels.

Still other devices have a different configuration in which the deviceincludes a plurality of reaction chambers disposed along the flowchannel and in fluid communication with the flow channel, with eachreaction chamber located within one of the temperature regions. Devicesof this type can also include a plurality of control channels, eachformed within an elastomeric material and separated from one of thereaction chambers by an elastomeric membrane, the membrane beingdeflectable into one of the reaction chambers in response to anactuation force applied to the control channel. As a result of suchactuation, sample can be transported between the reaction chambers. Insome instances, the plurality of reaction chambers are in fluidcommunication such that substantially all of the sample within theplurality of reaction chambers is collected at one of the plurality ofreaction chambers upon actuation of the control channels associated withthe other reaction chambers.

Other microfluidic devices that are provided herein include (a) asubstantially circular microfabricated flow channel in fluidcommunication with an inlet; (b) a plurality of temperature regions,each region located at a different location along the substantiallycircular flow channel; and (c) a temperature controller operativelydisposed to regulate the temperature within at least one of theplurality of temperature regions.

Methods utilizing devices of the foregoing design are also providedherein. Such methods generally involve providing a microfluidic devicesuch as described above, introducing a sample into the flow channels,and then transporting the sample between the different temperatureregions. Such methods can involve introducing a nucleic acid sample andcomponents for conducting a nucleic acid amplification reaction into theflow channel. The sample and the components for the amplificationreaction are then repeatedly cycled through the flow channel such thatthe sample and components are exposed to the temperature regionsmultiple times and an amplified product is formed. The methods canfurther involve detection of the amplified product. In some methods, theamplified product bears a detectable label and detection involvesdetecting the label. Detection in other methods involves contacting theamplified product with a label such that the amplified product becomeslabeled. Exemplary labels suitable for such methods includeinterchelating dyes and molecular beacons. Still other detection schemesinvolve conducting a quantitative PCR assay, detecting amplified productby gel electrophoresis, or measuring capacitance of a solutioncontaining the amplified product.

Microfluidic devices of the type described above can also be utilized toconduct sequencing reactions. In such methods a nucleic acid sample isintroduced into the flow channel together with one or more componentsrequired for conducting a sequencing reaction. Quantitative PCR can alsobe performed with the devices. Such methods involve introducing anucleic acid and one or more components for conducting a quantitativePCR reaction into the flow channel and then transporting the sample andcomponents through the different temperature regions.

Other microfluidic devices that are disclosed herein have a differentdesign and are in the form of an array or matrix of junctions orreaction chambers located at the intersection of horizontal and verticalflow channels. Microfluidic devices of this type enable a large numberof reactions to be conducted simultaneously. Certain of these devicesinclude (a) a substrate comprising an elastomeric material; (b) a firstplurality of flow channels formed within the substrate; (c) a secondplurality of flow channels, each formed within the substrate and influid communication with an inlet, the second flow channels intersectingthe first flow channels to define an array of reaction chambers; (d)isolation valves selectively actuatable to block flow between junctionsalong at least one of the first and second flow channels, and toregulate solution flow to the reaction chambers; (e) a plurality oftemperature regions located along each of the second plurality ofmicrofabricated flow channels; and (f) a temperature controlleroperatively disposed to regulate temperature at one or more of thetemperature regions. In certain of these devices, the isolation valvescomprise a first and second valve that have differing activationthresholds.

Other devices include (a) a substrate comprising an elastomericmaterial; (b) a first plurality of flow channels formed within thesubstrate; (c) a second plurality of flow channels, each formed withinthe substrate and in fluid communication with an inlet, the second flowchannels intersecting the first flow channels to define an array ofreaction chambers; and (d) isolation valves selectively actuatable toblock flow between junctions along at least one of the first and secondflow channels, and to regulate solution flow to the reaction chambers,wherein the isolation valves comprise a first and second isolation valvethat have differing activation thresholds. In certain devices, reactionchambers are separated from one another by at least two first isolationvalves or at least two second isolation valves.

With array-based microfluidic devices such as just described, theisolation valves can comprise an elastomeric membrane that separates acontrol channel and the microfabricated flow channel upon which thevalve acts, the elastomeric membrane able to be deflected into orrefracted from the flow channel upon which it acts in response to anactuation force. Devices of this design can also include one or morepumps to flow solution through the horizontal and/or vertical flowchannels. Certain of these pumps comprise one or more control channels,each of the control channels formed within an elastomeric material andseparated from one of the horizontal or vertical flow channels by asection of an elastomeric membrane, the membrane being deflectable intoor retractable from the horizontal or vertical flow channel upon whichit acts in response to an acutation force applied to the controlchannel.

Array-based devices such as the foregoing can be utilized to conductdiverse types of analyses, including nucleic acid amplificationreactions, synthesis reactions and screening analyses. In general suchmethods involve (a) providing an array-based device such as describedabove, (b) introducing a sample and one or more reactants into thereaction chambers by selective actuation of one or more of the isolationvalves, whereby reaction between the sample and the one or morereactants occurs, and (c) heating regions of the microfluidic device topromote reaction between the sample and the one or more reactants withinthe reaction chambers. In certain methods, the sample and the one ormore reactants are introduced into the first and second plurality offlow channels, and then the isolation valves actuated to allow thesample and the one or more reactants to mix by diffusion within thereaction chambers.

When nucleic acid amplification reactions are performed, the sample is anucleic acid containing sample and the one or more reactants arereactants required for the particular type of amplification reaction.The heating step promotes reaction between the nucleic acid and the oneor more reactants to form an amplified product. The resulting amplifiedproduct can be detected according to the methods described above.

Certain methods involve introducing samples and/or one or more of thereactants under pressure. Some of these methods involve positioning themicrofluidic device in a holder that is configured to form an air tightchamber over an inlet to each of the first plurality of flow channels.Sample and/or one or more of the reactants are placed into the inlets tothe first plurality of flow channels. The airtight chamber is thenpressurized, thereby forcing the sample and/or one or more reactantsinto the flow channels.

Nucleic acid amplification reactions utilizing array-based devicesgenerally involve introducing nucleic acid samples into each of thefirst plurality of flow channels and one or more reactants forconducting an amplification reaction into each of the second pluralityof flow channels, whereby the nucleic acid samples and the one or morereactants become mixed. By heating regions of the microfluidic device topromote reaction, amplified product can be formed. Amplified product canbe detected according to the methods set forth above. Sequencing andquantitative PCR reactions can be performed in related fashion.

Temperature is controlled at the temperature regions or reactionchambers of the microfluidic devices utilizing any of a number oftemperature controller including, but not limited to, a Peltier device,a resitive heater, a heat exchanger and an indium tin oxide element.Certain temperature controllers suitable for use with the array-basedmicrofluidic devices are provided. Some of these controllers include (a)a plate assembly comprising a first plate and a second plate that areseparated from one another by a separation material, the separationmaterial forming a fluid-tight seal around the periphery of the plates,the space between the plates and bounded by the separation materialdefining a chamber; (b) an inlet assembly located at a first end regionof the plate assembly and in fluid communication with the chamber; and(c) an outlet assembly located at a second end region of the plateassembly opposite the first end region and in fluid communication withthe chamber such that fluid in the chamber can exit therefrom via theoutlet assembly. With controllers of this design a region locatedbetween the inlet and outlet assembly and adjacent or abutting a surfaceof the first plate is adapted to receive a microfluidic chip, the firstand second plate comprise a transparent region that permits opticaldetection of the microfluidic chip, and the top plate is less than 100microns thick. Other temperature controllers include a plate assemblythat comprises (i) a hinged assembly that comprises a first plate and asecond plate that are hingeably connected, such that the first plate canbe moved toward or away from an upper face of the second plate; (ii) athird plate; (iii) a separation material that separates a lower face ofthe second plate opposite the upper face from the third plate and formsa fluid-tight seal therebetween; (iv) a chamber formed by the spacebetween the lower face of the second plate and the third plate and beingbounded by the separation material; and (v) a pair of holes, one holebeing located in the first plate and the other hole being located in thesecond plate, the holes positioned such that when the first plate isfolded onto the second plate, the pair of holes are aligned. In additionto the plate assembly, the temperature controller includes an inletassembly located at a first end region of the plate assembly and influid communication with the chamber; and (c) an outlet assembly locatedat a second end region of the plate assembly opposite the first endregion and in fluid communication with the chamber such that fluid inthe chamber can exit therefrom via the outlet assembly, wherein thehinged assembly is adapted to receive a microfluidic chip between thefirst and second plate.

To facilitate amplification reactions, polymerase can be immobolizedonly within temperature regions at which primer extension occurs. Thismeans that non-thermophilic polymerases can be utilized as thepolymerase is not exposed to the higher temperatures required toseparate the template nucleic acid from the extension product.

In certain devices, detection is facilitated by immobilizing one or morenucleic acids to a region of a flow channel or reaction chamber. Suchnucleic acids can serve as probes to bind selected amplificationproducts, for example. By spatially depositing the nucleic acids inknown locations, the presence or absence of particular target nucleicacids can be ascertained according to the location at which a targetnucleic acid binds to the array of nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of an exemplary microfluidic devicefor conducting thermal cycling reactions that utilizes a substantiallycircular flow channel configuration.

FIG. 2 is a schematic representation of another exemplary microfluidicdevice for conducting thermal cycling reactions in which the flowchannel incorporates a plurality of reaction chambers.

FIGS. 3A and 3B are cross-sectional views of an unactuated and actuatedreaction chamber such as present in the microfluidic device illustratedin FIG. 2.

FIG. 4 is a cross-sectional view showing the location of a heaterrelative to the flow channel in a temperature region of a microfluidicdevice.

FIG. 5 is a plan view of a structure for high throughput screening ofnucleic acids in accordance with one embodiment of the presentinvention.

FIGS. 6A-6C are enlarged views of a portion of the structure of FIG. 5showing its operation.

FIG. 7A is a schematic representation of an exemplary microfluidicdevice comprising a rotary channel, pumps and heaters. Three heatersarranged in the following order (clockwise from upper right):denaturation, annealing, and extension. (In the 2-step Taqman PCR, onlythe two heaters for denaturation and extension were used.)

FIG. 7B is a schematic representation of assembly of the rotarymicrochip and the heaters showing control layer, flow layer, glasscover-slip and heaters, with arrows indicating the air and fluid vias(i.e., the openings in the layer which connect to a flow or controlchannel).

FIG. 8 is a graph showing bulk confirmation of the Taqman amplificationof the human β-actin gene fragment.

FIGS. 9A and 9B show gel electrophoresis analysis of bulk PCR products.

FIG. 10 is a graph of fluorescence increase in a spatially cycled Taqmanassay performed using a microfluidic device as provided herein.

FIG. 11A shows a bar graph of results of three-temperature spatialcycling PCR of a segment of λ DNA.

FIG. 11B shows a bar graph of results of three-temperature temporalcycling PCR of a segment of λ DNA.

FIG. 12 is a plan view of an exemplary matrix or array microfluidicdevice that can be utilized to conduct nucleic acid amplificationreactions and other types of reactions.

FIGS. 13A-13D are enlarged views of a portion of the device of FIG. 12showing its operation.

FIGS. 14A-14C are enlarged views of a portion of the device of FIG. 12showing its operation.

FIGS. 15A-15D show plan and cross-sectional views of an exemplarytemperature controller for use with various microfluidic devices such asthose disclosed herein.

FIG. 16 illustrates a exemplary holder which forms an airtight chamberover wells or recesses in a microfluidic device, thus allowing forpressurized introduction of solutions into the microfluidic device.

FIG. 17 illustrate another exemplary temperature controller that can beutilized to regulate temperature within a microfluidic device such asthose disclosed herein.

FIG. 18 shows an “S” shaped control channel design that can provideperistaltic pumping action on an underlying flow channel.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

A “flow channel” refers generally to a flow path through which asolution can flow.

The term “valve” unless otherwise indicted refers to a configuration inwhich a flow channel and a control channel intersect and are separatedby an elastomeric membrane that can be deflected into or retracted fromthe flow channel in response to an actuation force.

The term “elastomer” and “elastomeric” has its general meaning as usedin the art. Thus, for example, Allcock et al. (Contemporary PolymerChemistry, 2nd Ed.) describes elastomers in general as polymers existingat a temperature between their glass transition temperature andliquefaction temperature. Elastomeric materials exhibit elasticproperties because the polymer chains readily undergo torsional motionto permit uncoiling of the backbone chains in response to a force, withthe backbone chains recoiling to assume the prior shape in the absenceof the force. In general, elastomers deform when force is applied, butthen return to their original shape when the force is removed. Theelasticity exhibited by elastomeric materials can be characterized by aYoung's modulus. The elastomeric materials utilized in the microfluidicdevices disclosed herein typically have a Young's modulus of betweenabout 1 Pa-1 TPa, in other instances between about 10 Pa-100 GPa, instill other instances between about 20 Pa-1 GPa, in yet other instancesbetween about 50 Pa-10 MPa, and in certain instances between about 100Pa-1 MPa. Elastomeric materials having a Young's modulus outside ofthese ranges can also be utilized depending upon the needs of aparticular application.

Some of the microfluidic devices described herein are fabricated from anelastomeric polymer such as GE RTV 615 (formulation), a vinyl-silanecrosslinked (type) silicone elastomer (family). However, the presentmicrofluidic systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. Given the tremendous diversity of polymer chemistries,precursors, synthetic methods, reaction conditions, and potentialadditives, there are a large number of possible elastomer systems thatcan be used to make monolithic elastomeric microvalves and pumps. Thechoice of materials typically depends upon the particular materialproperties (e.g., solvent resistance, stiffness, gas permeability,and/or temperature stability) required for the application beingconducted. Additional details regarding the type of elastomericmaterials that can be used in the manufacture of the components of themicrofluidic devices disclosed herein are set forth in U.S. applicationSer. No. 09/605,520, and PCT Application No. 00/17740, both of which areincorporated herein by reference in their entirety.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused herein to include a polymeric form of nucleotides of any length,including, but not limited to, ribonucleotides or deoxyribonucleotides.There is no intended distinction in length between these terms. Further,these terms refer only to the primary structure of the molecule. Thus,in certain embodiments these terms can include triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. They also include modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,”include polydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), any other type ofpolynucleotide which is an N- or C-glycoside of a purine or pyrimidinebase, and other polymers containing normucleotidic backbones, forexample, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA.

A “probe” is an nucleic acid capable of binding to a target nucleic acidof complementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation, thus forming a duplex structure. The probe binds orhybridizes to a “probe binding site.” The probe can be labeled with adetectable label to permit facile detection of the probe, particularlyonce the probe has hybridized to its complementary target. The labelattached to the probe can include any of a variety of different labelsknown in the art that can be detected by chemical or physical means, forexample. Suitable labels that can be attached to probes include, but arenot limited to, radioisotopes, fluorophores, chromophores, mass labels,electron dense particles, magnetic particles, spin labels, moleculesthat emit chemiluminescence, electrochemically active molecules,enzymes, cofactors, and enzyme substrates. Probes can vary significantlyin size. Some probes are relatively short. Generally, probes are atleast 7 to 15 nucleotides in length. Other probes are at least 20, 30 or40 nucleotides long. Still other probes are somewhat longer, being atleast 50, 60, 70, 80, 90 nucleotides long. Yet other probes are longerstill, and are at least 100, 150, 200 or more nucleotides long. Probescan be of any specific length that falls within the foregoing ranges aswell.

A “primer” is a single-stranded polynucleotide capable of acting as apoint of initiation of template-directed DNA synthesis under appropriateconditions (i.e., in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, DNA or RNApolymerase or reverse transcriptase) in an appropriate buffer and at asuitable temperature. The appropriate length of a primer depends on theintended use of the primer but typically is at least 7 nucleotides longand, more typically range from 10 to 30 nucleotides in length. Otherprimers can be somewhat longer such as 30 to 50 nucleotides long. Shortprimer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. A primer neednot reflect the exact sequence of the template but must be sufficientlycomplementary to hybridize with a template. The term “primer site” or“primer binding site” refers to the segment of the target DNA to which aprimer hybridizes. The term “primer pair” means a set of primersincluding a 5′ “upstream primer” that hybridizes with the complement ofthe 5′ end of the DNA sequence to be amplified and a 3′ “downstreamprimer” that hybridizes with the 3′ end of the sequence to be amplified.

A primer that is “perfectly complementary” has a sequence fullycomplementary across the entire length of the primer and has nomismatches. The primer is typically perfectly complementary to a portion(subsequence) of a target sequence. A “mismatch” refers to a site atwhich the nucleotide in the primer and the nucleotide in the targetnucleic acid with which it is aligned are not complementary. The term“substantially complementary” when used in reference to a primer meansthat a primer is not perfectly complementary to its target sequence;instead, the primer is only sufficiently complementary to hybridizeselectively to its respective strand at the desired primer-binding site.

The term “complementary” means that one nucleic acid is identical to, orhybridizes selectively to, another nucleic acid molecule. Selectivity ofhybridization exists when hybridization occurs that is more selectivethan total lack of specificity. Typically, selective hybridization willoccur when there is at least about 55% identity over a stretch of atleast 14-25 nucleotides, preferably at least 65%, more preferably atleast 75%, and most preferably at least 90%. Preferably, one nucleicacid hybridizes specifically to the other nucleic acid. See M. Kanehisa,Nucleic Acids Res. 12:203 (1984).

The term “label” refers to a molecule or an aspect of a molecule thatcan be detected by physical, chemical, electromagnetic and other relatedanalytical techniques. Examples of detectable labels that can beutilized include, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, enzymes linked to nucleic acidprobes and enzyme substrates. The term “detectably labeled” means thatan agent has been conjugated with a label or that an agent has someinherent characteristic (e.g., size, shape or color) that allows it tobe detected without having to be conjugated to a separate label.

II. Overview

A variety of microfluidic devices and methods for conducting analysesthat benefit from temperature control are provided herein. Certaindevices generally include a microfabricated flow channel along which aplurality of different temperature regions are located. In some of thesedevices, the flow channel forms a loop to allow for continuous andcyclic solution flow through the flow channel, thus allowing a sample tobe repeatedly exposed to the different temperature regions.

Still other microfluidic devices provided herein comprise a plurality ofintersecting flow channels to form an array or matrix of reactionchambers or junctions at which reactions can occur. Such devices permita large number of reactions (e.g., nucleic acid amplification reactions)to be performed simultaneously in a multiplex format, or facilite highthroughput screening of samples (e.g., screening compounds that bind tonucleic acids or proteins). Temperature controllers can regulatetemperature for the entire device or a portion thereof (e.g., a junctionat which flow channels intersect). Some of the devices having such anarchitecture include valves that have different actuation thresholds. Byjudicious placement of such valves along a flow channel, certainreagents or sample can be segregated within a flow channel andsubsequently released for diffusive mixing with other reagents in theflow channel.

The devices are further characterized in part by including variouscomponents such as flow channels, control channels, valves and/or pumps,at least some of which are manufactured from elastomeric materials. Thisis in sharp contrast to conventional microfluidic devices that typicallyare based on silicon substrates (i.e., silicon chips). Additionally,with many of the devices disclosed herein, amplification reactions canbenefit from aspects of both space and time domain amplificationapproaches, whereas heretofore amplification reactions were typicallylimited to utilizing only one approach or the other. Moreover, asalluded to above, certain of the devices and methods utilize asubstantially circular flow channel through which samples and reactantsolutions can be repeatedly transported. With conventional microfluidicdevices that utilize electric fields to control solution flow,continuous circular flow is not possible.

The devices provided herein are useful in performing a variety ofanalyses requiring temperature control. This includes template extensionreactions that involve thermal cycling. Such template extensionreactions include both linear amplification reactions (extensionreactions conducted with a single primer) and exponential amplifications(extension reactions conducted with both forward and reverse primers).Thus, the term amplification refers to both linear amplificationreactions (e.g., certain sequencing reactions and single base pairextension reactions) as well as exponential amplification reactions(e.g., PCR).

Certain methods can be conducted in a multiplexing format in whichmultiple target nucleic acids are simultaneously amplified in a flowchannel as the targets and amplification agents are cycled through thedifferent temperature regions, or within a reaction chamber wheretemperature is cycled. Such reactions can be performed by utilizingprimers specifically complementary to the different targets and thenutilizing differential detection methods to detect different amplifiedproducts. Alternatively, multiple temperature cycling devices can beincorporated into a single device in which solution flow is controlledthrough common control channels.

III. General Device Structure

The microfluidic devices disclosed herein are typically constructed atleast in part from elastomeric materials and constructed by single andmultilayer soft lithography (MLSL) techniques and/or sacrificial-layerencapsulation methods (see, e.g., Unger et al. (2000) Science288:113-116, and PCT Publication WO 01/01025, both of which areincorporated by reference herein in their entirety for all purposes).Utilizing such methods, microfluidic devices can be designed in whichsolution flow through flow channels of the device is controlled, atleast in part, with one or more control channels that are separated fromthe flow channel by an elastomeric membrane or segment. This membrane orsegment can be deflected into or refracted from the flow channel withwhich a control channel is associated by applying an actuation force tothe control channels. By controlling the degree to which the membrane isdeflected into or retracted out from the flow channel, one can slow orentirely block solution flow through the flow channel. Usingcombinations of control and flow channels of this type, one can preparea variety of different types of valves and pumps for regulating solutionflow.

The devices and methods described herein utilize such valves and pumpsto control solution flow through a series of reaction chambers at whichtemperature can be regulated, and/or a series of different temperaturezones or regions that are disposed along one or more flow channels,thereby providing a way to tailor a temperature sequence or profileaccording to the particular temperature requirements of an analysis. Asnoted supra, such devices can be utilized in a variety of analyses thatutilize thermocycling, particularly nucleic acid amplification processessuch as PCR, for example.

More specifically, some of the microfluidic devices that are providedtypically include a flow channel into which a sample can be introducedand transported. In certain designs, the flow channel forms a loop suchthat sample introduced into the loop can be cycled multiple timesthrough the enclosed loop. Such a flow channel can be referred to as arotary microfluidic flow channel or simply rotary flow channel. The loopcan be substantially circular in certain devices but other shapes suchas rectangular, triangular, hexagonal, octogonal, or any other suitablegeometrical shape, can be utilized as well.

A plurality of temperature zones or regions are spaced along the flowchannel. These temperature regions are regions of the loop in which atemperature controller regulates temperature. These regions can becontrolled by a single temperature controller but, more typically, thetemperature in each region is regulated by a separate temperaturecontroller. The temperature regions can be of varying lengths as a wayto further control the temperature of the solution flowing through theflow channel. With certain devices, the width and/or depth of the flowchannel is varied in the different temperature zones to provide afurther mechanism for regulating temperature.

As noted above, solution flow through the flow channel is controlledusing one or more control channels that are separated from the flowchannel by a semipermeable membrane. The deflection of such membranesinto the flow channel can be used to block solution flow (i.e., as avalve), to expel solution from one region of the flow channel to anotherregion (i.e., as a pump), and, by staggering the time at which a seriesof control channels are actuated so as to stagger membrane deflectioninto the flow channel, to produce a peristaltic pumping action. With theuse of such arrangements, one can regulate solution flow through theflow channel without having to utilize electric fields to effectuatetransport, although such techniques can also be utilized in certainapplications.

Certain devices also include a detection section at which unreactedagents and/or products can be detected. This detection section caninclude detectors that are incorporated into the device or be alignedwith a detector that is not incorporated into the device. In someinstances, the detection section includes the flow channel in which thethermal cycling reaction takes place. In other designs, the detectionsection is located at another part of the device, typically downstreamfrom an outlet connected to the flow channel in which thermal cyclingoccurs. Because the microfluidic devices provided herein can be made ofelastomers that are substantially optically transparent, the devices canbe used with certain detection systems that cannot be utilized withconventional devices manufactured from silicon.

Thus, in operation, certain methods involve introducing a sample intothe flow channel and then actuating control channels associated with theflow channel to move the sample from one temperature region to another.With certain devices, solution can be transported semi-continuously orcontinuously around the looped flow channel such that the sample isrepeatedly exposed to the different temperature zones in a cyclicfashion.

For applications involving nucleic acid template extension reactionssuch as nucleic acid amplification reactions, a sample containing anucleic acid target is introduced into the flow channel. Components forconducting the amplification (e.g., primers, deoxynucleotides,polymerase, buffers and cofactors such as metal ions) are alsointroduced into the flow channel. The order in which these solutions areadded can be reversed. The temperature within the temperature regions isselected to promote the major processes involved in amplificationreactions, namely annealing of primer(s) to the target nucleic acid,extension of the primers, dissociation of complementary strands of theextension product, and then reannealing of primer to target nucleic acidor amplified product.

Other microfluidic devices include some of the foregoing features butdiffer in that rather than having a loop-shaped flow channel, the deviceincludes a plurality of intersecting horizontal and vertical flowchannels, the intersections providing reaction chambers at whichreactions can occur. Temperature at the reaction chambers or junctionscan be controlled by a temperature controller that regulates temperaturethroughout the device, or one or more temperature controllers thatregulate temperature at one or more of the reaction chambers.

In operation, these array or matrix type devices involve flowing a setof reagents through the horizontal flow channels and one or more sets ofreagents through the vertical flow channels. These reagents become mixedtogether within the junctions of the horizontal and vertical flowchannels. Typically, products and/or unreacted regents are detected atthe junctions of the flow channels or at regions of the flow channeladjacent the junctions.

The following sections describe in greater detail a number of specificconfigurations that can be utilized to achieve temperature controlduring analyses, particularly nucleic acid amplification reactions. Itshould be understood, however, that these configurations are exemplaryand that modifications of these systems will be apparent to thoseskilled in the art.

IV. Devices Utilizing a Substantially Circular Flow ChannelConfiguration

A. Architecture

Devices of this configuration utilize a rotary pump design in which oneor more elastomeric pump(s) act to circulate fluid through asubstantially circular flow channel. As used herein the term“substantially circular” has the meaning known in the art and refers toconfigurations that are circles, as well as variations from a circularconfiguration such as ellipsoids, ovals and octagons for example.

Devices of this design can be manufactured by multilayer softlithography. Such devices generally consists of two layers that arejoined together: a first layer into which flow channels are formed andthrough which sample and reactants flow, and a second layer into whichcontrol channels are formed. These two layers are joined such that thecontrol channels are appropriately oriented with respect to the flowchannels. The resulting device is typically then affixed to a substrate(e.g., a glass slide) such that the substrate forms one wall of the flowchannel. However, a thin elastomeric membrane can be placed over theexposed flow channel to form a device in which the flow channel isentirely formed of elastomer.

One exemplary microfluidic device for conducting thermal cyclingreactions is illustrated in FIG. 1. This device 100 includes in thelayer with the flow channels a plurality of sample inputs 102 a and 102b, a mixing T-junction 104, a central circulation loop 106 (i.e., thesubstantially circular flow channel), and an output channel 108. Asindicated supra, the intersection of a control channel with a flowchannel can form a microvalve. This is so because the control and flowchannels are separated by a thin elastomeric membrane that can bedeflected into the flow channel or retracted therefrom. Deflection orretraction of the elastomeric membrane is achieved by generating a forcethat causes the deflection or retraction to occur. In certain systems,this is accomplished by increasing or decreasing pressure in the controlchannel as compared to the flow channel with which the control channelintersects. However, a wide variety of other approaches can be utilizedto actuate the valves including various electrostatic, magnetic,electrolytic and electrokinetic approaches. Such approaches aredescribed, for example, in PCT Publication WO 01/01025, previouslyincorporated herein in its entirety for all purposes.

The substantially circular central loop 106 and the control channels 116that intersect with it form the central part of the rotary pump. Thepump(s) 114 a and 114 b that cause solution to be flowed through thesubstantially circular flow channel 106 consist of a set of at leastthree control channels 116 that are adjacent to one another and whichintersect the substantially circular branch flow channel 106 (i.e., thecentral loop). When a series of on/off actuation sequences, such a 001,011, 010, 110, 100, 101, are applied to the control channels 116, thefluid in the central loop 106 can be peristaltically pumped in a chosendirection, either clockwise or counterclockwise. The peristaltic pumpingaction results from the sequential deflection of the membranesseparating the control channels 116 and flow channel 106 into or out ofthe flow channel. In general, the higher the actuation frequency, thefaster the fluid rotates through the central loop. However, a point ofsaturation is eventually reached at which increased frequency does notresult in faster fluid flow. This is primarily due to limitations in therate at which the membrane can return to an unactuated position. Whilethe system shown in FIG. 1 shows two sets of pumps 114 a and 114 b(i.e., two sets of three control channels 116 that overlay thesubstantially circular flow channel 106) a single pump can be utilized(i.e., a single set of three control channels overlaying thesubstantially circular flow channel). Furthermore, while each pump isshown as including three control channels, more control channels can beutilized. It should also be understood that the three control channelscan be different segments of a single control channel that overlay theflow channel.

An example of such an arrangement is an “S” shaped control channel thatpasses over the flow channel at least three times. FIG. 18 presents aplan view of a pump structure, wherein serpentine control channel 1800crosses over underlying flow channel 1802 at three different points,thereby creating three valve structures 1804, 1806, and 1808 havingmembranes 1804 a, 1806 a, and 1808 a respectively. Application of a highpressure signal to first end 1800 a of control channel 1800 causes ahigh pressure signal to propagate to first valve 1804, then to secondvalve 1806, and finally to third valve 1808, as shown in TABLE 1.

TABLE 1 TIME VALVE 7804 VALVE 7806 VALVE 7808 T₀ 1 0 0 T₁ 1 1 0 T₂ 1 1 1T₃ 0 1 1 T₄ 0 0 1 T₅ 0 0 0 0 = valve open; 1 = valve closed

The resulting sequence of deflection of membranes 1804 a, 1806 a, and1808 a creates a pumping action in flow channel 1802 in direction F.Moreover, relaxation of pressure at first end 1800 d of control channel1800 at time T₃ would cause a low pressure signal to propagate to firstvalve 1804, then to second valve 1806, and finally to third valve 1808,setting the stage for another pumping sequence without causing reversalin direction of material previously flowed through channel 1802.

A variety of different auxiliary flow channels which are in fluidcommunication with the central loop 106 can be utilized to introduce andwithdrawn sample and reactant solutions from the central loop. Asdepicted in FIG. 1, for example, a plurality of inlets 102 a and 102 bthat are joined to a T-shaped flow channel 104 that is in fluidcommunication with the central loop 106 can be used to introduce sampleand solutions containing reactants or assay components into the centralloop 106. Similarly, one or more exit or outlet flow channels 108 influid communication with central loop 106 can be utilized to removesolution from central loop 106. Control valves 110 and ‘112 can beutilized at the inlet(s) and the outlet(s), respectively, to preventsolution flow into or out from the central loop 106.

With continued reference to FIG. 1, it can be seen that a plurality oftemperature regions 118, 120 and 122 are located at different locationsalong the central loop 106. As shown, these temperature regions 118, 120and 122 can be of varying lengths. In this way, one can flow solutionthrough the central loop at a continuous flow rate but still vary thelength of time that solution is exposed to a particular temperature. Asdescribed in greater detail infra, a variety of different types oftemperature controllers can be utilized to regulate temperature withinthe different temperature regions. In certain designs, a heater (e.g.,sputtered resistive metal) is positioned between the flow channel andthe substrate (see FIG. 4). Usually the temperature is different in eachof the different temperature regions. While the device shown in FIG. 1shows three separate temperature regions, it should be understood thatfewer or more temperature regions can be utilized. The number of regionsdepends primarily on the nature of the reaction and the size of thedevice. A related device described in the examples below describe asystem with just two temperature regions.

Flow channel sizes and shapes can vary. With certain devices, thediameter of the channel tends to range from about 1 mm to 2 cm, althoughthe diameter can be considerably larger in certain devices (e.g., 4, 6,8, or 10 cm). Limits on how small the diameter of the circular flowchannel can be are primarily a function of the limits imposed by themultilayer soft lithography processes. Channel widths (either flow orcontrol) usually vary between 30 μm and 250 μm. However, channel widthin some devices is as narrow as 1 μm. Channels of larger widths can alsobe utilized, but generally require some type of structural supportwithin the flow channel. Channel height generally varies between 5 and50 μm. The flow channel is typically rounded to allow for completeblockage of the channel once the membrane is deflected into the channel.In some devices, the channels have shapes such as octagons or hexagons.In certain devices, the flow channels are rounded and 100 μm wide and 10μm high and control channels are 100 μm wide and 10 μm high. One systemthat has been utilized in certain studies has utilized a central loophaving a diameter of 2 cm, a flow channel depth of 100 μm and a width of10 μm. While the channels typically have the foregoing sizes and shapes,it should be recognized that the devices provided herein are not limitedto these particular sizes and shapes.

Devices utilizing a substantially circular design allow considerableflexibility in exposing samples to desired temperature profiles becauseof the ability to control temperature in both the time and spacedomains. With the central loop and the integrated pumps, these devicesalso permit one to continuously and repeatedly to flow solution throughthe different temperature zones. However, by periodically altering therate of control valve actuation, one can also regulate how long solutionis exposed to any particular temperature.

B. Methods for Conducting Analyses

A wide variety of applications can be conducted with devices 100 havingthe design shown in FIG. 1, especially applications requiringtemperature cycling. Before introducing solution into device 100, it issometimes helpful to first purge the flow channels within the device ofair. This avoids potential complications resulting from the formation ofair bubbles within the flow channels that can disrupt solution flowthrough the flow channels. Because the devices described herein arewholly or largely manufactured from elastomeric compounds that have acertain degree of porosity, this can conveniently be achieved bypressurizing the device which causes any gas to diffuse through thepores of the device.

Once device 100 has been primed, sample and any necessary reagents arecollectively or separately introduced into the central loop 106 via theinlets 102 a and 102 b. The control valves 110 positioned adjacent theinlets 102 a and 102 b and the T intersection 104 are initially openedto allow solution to flow into the central loop 106. After the desiredsamples and reagents have been introduced into the central loop 106, thecontrol valves 110 at the inlets 102 a and 102 b and outlet 108 areclosed. Solution is circulated through the central loop 106 by actuatingthe pumps 114 a and 114 b. As the solution is cycled around the centralloop 106, the sample and reagents are exposed to a sequence of differenttemperatures.

Following completion of the temperature cycling, the presence ofremaining reactants and/or products generated during reaction can bedetected. With certain devices detection is accomplished by detectingremaining reactants and/or products within the central loop 106 itself.Thus, the rotary pump 106 constitutes a detection region. In otherinstances, the control valve 112 of the outlet 108 is opened so solutioncan be removed from the central loop 106. The solution that is withdrawncan then be transported via a flow channel in fluid communication withthe outlet 108 to a detection region elsewhere on the microfluidicdevice. Alternatively, the solution can be removed from the microfluidicdevice and analyzed on a separate device.

As described in greater detail below, a wide variety of detectionoptions can be utilized to detect the reactants and/or products. Theparticular method employed depends upon the nature of the reactantand/or product being detected.

In the specific instance of nucleic acid amplification reactions, asample containing or potentially containing a target nucleic acid isintroduced into the central loop 106 via one of the inlets 102 a and 102b. The other reagents necessary to conduct the amplification reactionare similarly introduced via one of the inlets 102 a and 102 b. Thesample and reagents can be introduced collectively or separately.Reagents typically utilized to conduct an amplification reaction willvary somewhat depending upon the particular type of amplificationreaction being conducted and are known to those of ordinary skill in theart. Typical reagents include a primer or primers (e.g., forward andreverse primers) that specifically hybridize to the target nucleic acid,the four deoxynucleoside triphosphates (i.e., dATP, dTTP, dGTP anddCTP), a polymerase, a buffer and various other cofactors required bythe polymerase (e.g., metal ion).

Following introduction of the sample and the necessary amplificationreagents into the central loop 106, the sample and amplificationreagents are circulated around the central loop 106 under the action ofthe pumps 114 a and 114 b. The number of temperature regions 118, 120and 122 and the temperature therein can be selected according to theparticular amplification reaction being conducted. The device shown inFIG. 1 is useful for conducting standard PCR reactions as it includesthree temperature zones 118, 120 and 122 to promote the three primaryevents that occur during a PCR amplification, namely: (1) annealing ofthe primer(s) to its/their complementary target, (2) extension of theprimer as the polymerase enzyme incorporates additional nucleotides, and(3) denaturation of the double stranded nucleic acid such that theprimer(s) can reanneal to the target or a newly replicated strand.Temperatures typical for conducting PCR reactions are 95° C.(denaturation), 72° C. (extension) and 49-69° C. (annealing).

C. Variations

In some applications, the solution flow rate is varied. One approach forachieving this is to alter the rate at which the control channels areactuated. Alternatively, actuation of the control channels can be keptconstant but the dimensions of the flow channel altered at differentregions. Thus, the pumping rate can be kept constant while solution flowthrough the flow channel can be varied depending upon the size of theflow channel. Thus, in certain devices, the flow channel dimension canvary between one or more of the temperature regions as a way to provideanother level of temperature control.

During the thermal cycling process, various reagents or solutions canoptionally be introduced into the central loop via the inlets. Forexample, additional polymerase can be introduced if the enzyme starts tobecome denatured during the cycling process. More buffer solution can beintroduced to compensate for solution loss due to evaporation.Similarly, solution can be withdrawn via the outlet during the cyclingprocess as part of time course studies or to monitor the progress of thereaction, for example.

V. Devices Utilizing a Plurality of Reaction Chambers

A. Architecture

An example of another configuration that can be utilized inthermocycling reactions is illustrated in FIG. 2. As with themicrofluidic device illustrated in FIG. 1, this device 200 also iscomposed of two layers that are joined together: a first layer intowhich flow channels are formed and through which sample and reactantsflow, and a second layer into which control channels are formed. Afterthese two layers are joined such that the control channels properlyintersect with the appropriate flow channels, the resulting device istypically affixed to a support (e.g., glass). Consistent with thearchitecture of the device shown in FIG. 1, the layer with the flowchannels has a plurality of sample inputs 202 a and 202 b, a mixingT-junction 204, and an output channel 206 (see FIG. 2). This device,however, differs in that a plurality of reaction chambers 208 a, 208 band 208 c are disposed along the primary flow channel 210. In general, adifferent reaction chamber is provided for each different temperatureregion that is needed to conduct the analysis of interest.

The plurality of reaction chambers 208 a, 208 b and 208 c are in fluidcommunication with other reaction chambers in the system. The reactionchambers 208 a, 208 b, and 208 c are also operatively disposed withrespect to a control channel 212 a, 212 b and 212 c, respectively.Actuation of the control channel associated with a reaction chambercauses the solution within the chamber to be expelled. As depicted inFIGS. 3A and 3B, because the control channel 304 and reaction chamber308 are formed within an elastomeric material 302 (that is attached to asubstrate 310) and separated by a flexible elastomeric membrane 312,actuation of the control channel 304 results in deflection of themembrane 312 into the reaction chamber 306. Because the membrane 312 canmold to the shape of the reaction chamber 306, essentially all solutionand reactants 308 within the reaction chamber 306 is forced out of theactuated reaction chamber and into an unactuated reaction chamber.Reaction chambers 306 whose associated control channel 304 is actuatedare sometimes referred to simply as actuated chambers; conversely,reaction chambers 306 whose control channels 304 are not actuated arereferred to as unactuated reaction chambers. The reaction chambers incertain devices are fluidly connected such that actuation of all thecontrol channels but one results in substantially all of the solution inthe actuated reaction chambers being forced into the one reactionchamber whose control channel is not actuated (i.e., the sole unactuatedreaction chamber).

The flow channel can include as few as two reaction chambers. However,the number of reaction chambers can vary significantly and is limitedprimarily by the size of the microfluidic device. As noted supra, ingeneral the number of reaction chambers incorporated into the device isselected to correspond to the number of different temperature regionsone requires for an analysis. With many nucleic acid amplificationreactions three reaction chambers are utilized to accommodate thetemperatures for performing annealing, extension and denaturation.However, certain devices include 4, 5, 6, 7, 8, 9 or 10 reactionchambers. Other designs utilize tens or even hundreds of reactionchambers, for example.

With systems of this design a sample and/or reagents can be rapidlypassed back and forth between two or more reaction chambers by selectiveand alternate actuation of the corresponding control channels, thusresulting in the folding of a solution back onto itself. In this way,solutions can be rapidly mixed. Such configurations can also be utilizedin a variety of thermal cycling applications by repeatedly passing afluid through an established thermal gradient.

These devices allow reactions to be conducted with ultra small reactionvolumes because one can consolidate the different reaction chambers intoa small space and because solution is fully transferred between chambersdue to the flexibility of the membrane that causes transport. The highcompliance of the membrane can also accommodate for volume changesassociated with the thermocycling process. High speed cycling can beachieved because there is no need to ramp the temperature to the desiredlevel.

In the case of the particular device shown in FIG. 2, the device 200includes an inlet system 201 having one or more inlets 202 a and 202 band a mixing T junction 204 in fluid communication with the reactionchamber 208 a set to the first temperature in the temperature gradient.Control valves 214 in the inlet system 201 can be used to controlintroduction of the sample and other reagents. Device 200 includes threereaction chambers 208 a, 208 b and 208 c set to temperatures that areoptimal for the annealing, extension and denaturation processesassociated with the amplification reaction. Substantially all of thesolution within device 200 can be transported to the reaction chamberhaving the next temperature in the gradient by actuating all of theother reaction chambers. An outlet flow channel 206 is in fluidcommunication with one reaction chamber 208 c to allow reaction productsand unreacted reagents to be withdrawn. Typically, the outlet 206 is influid communication with the reaction chamber 208 c set to the finaltemperature in the temperature gradient.

B. Methods for Conducting Analyses

Sample and reagents are introduced into the first reaction chamber 208 ain the flow channel 210 via the inlets 202 a and 202 b. Prior tointroducing such solutions, the flow channel 210 and reaction chambers208 a, 208 b and 208 c can be purged of air as described supra to avoidpotential problems associated with air bubble formation. Onceintroduced, typically the sample and reagents are transported from onereaction chamber/temperature region to another by actuating the controlchannels of all of the reaction chambers, except for the reactionchamber into which solution is to be delivered. Thus, referring onceagain to FIG. 2, once sample and reagents have had sufficient exposureto the temperature of reaction chamber 208 a, substantially all of thesolution in each of the reaction chambers and other sections of the flowchannel system can be forced into reaction chamber 208 b by actuatingcontrol channels 212 a and 212 c associated with reaction chambers 208 aand 208 c, respectively. Similarly, solution can be forced into reactionchamber 208 c by actuating control channels 212 a and 212 b associatedwith reaction chambers 208 a and 208 b.

For nucleic acid amplifications, the general considerations set forthabove with respect to the device shown in FIG. 1 apply to methodsperformed with device 200 as well. The temperature of each reactionchamber is set for the temperature that promotes the annealing,extension and denaturation processes. Solution is transported betweenthe different reaction chambers by selectively actuating the appropriatecontrol channels as just described. Any amplified product formed andunreacted reagents can be transported out of the reaction chambers viathe outlet.

Detection of unreacted reagents and/or product can be conducted at oneor more than one of the reaction chambers. Alternatively, detection canbe conducted after reagents and product have been transported from thereaction chambers and at another location on the device. As with theother device, another option is to remove a portion of the withdrawnsolution and to analyze the withdrawn solution on another system.

C. Variations

The same general variations described supra in relation to the systemwith a substantially circular system also apply here.

VI. Devices Utilizing an Array of Reaction Chambers

A. First Exemplary Configuration

1. Architecture

Certain devices comprise a plurality of intersecting vertical andhorizontal flow channels which form an N×N array of cross-injectionjunctions or reaction chambers. A separate amplification reaction can beperformed at each junction, thereby allowing a very large number ofamplification reactions to be carried out on a single device at the sametime. The number of vertical and horizontal flow channels can varywidely and is limited primarily by the overall size of the microfluidicdevice and the size and spacing of the flow channels. Devices of thistype can include less than ten vertical and horizontal flow channelseach, but often include tens or even hundreds of vertical and horizontalflow channels to form a vary large number of cross-injection junctionsat which amplification reactions can take place.

FIG. 5 illustrates a specific example of such an alternative structurefor performing nucleic acid amplifications. The high throughputstructure of FIG. 5 comprises a five-by-five array 500 ofcross-injection junctions 502 formed by the intersection of parallelhorizontal flow channels 504 and parallel vertical flow channels 506.Array 500 enables the mixing of a nucleic acid sample S1-S5 with aprimer-containing solution P1-P5, for a total of 5×5=25 simultaneouspossible amplification reactions. The other components necessary forconducting a particular amplification reaction (e.g., polymerase,labeled and/or unlabeled nucleotides, metal ions and other cofactors)can be included in either the nucleic acid sample and/or with the primersolution.

Movement of nucleic acid sample solutions S1-S5 along horizontal flowchannels 504 is controlled in parallel by peristaltic pumps 508 formedby overlying control channels 510. Movement of primer solutions P1-P5along vertical flow channels 506 is controlled in parallel by peristaticpumps 512 formed by overlying control channels 514. Column valves 516and row valves 518 surround each junction 502 formed by the intersectionof horizontal and vertical flow lines 504 and 506.

Column valves 516 blocking flow in the vertical direction are controlledby a single control line 520. Row valves 510 blocking flow in thehorizontal direction are controlled a single control line 522. Forpurposes of illustration, only the first portion of control lines 520and 522 are shown in FIG. 5. However, it is to be understood that everyrow and column valve is controlled by these control lines.

During an amplification reaction, horizontal flow channels 504 introducesamples of five different nucleic acid samples (S1-S5) into junctions502, while vertical flow channels 506 introduce five differentprimer-containing solutions (P1-P5) to junctions 502. Through themetering technique described below in connection with FIGS. 6A-6C, all5×5=25 possible combinations of nucleic acid sample and primer arestored at the 5×5=25 junctions 502 of array 500. Each such amplificationreaction is mixed at a fixed ratio of S to P.

Once charged with the nucleic acid samples and primer solutions, themicrofluidic array is then cycled through temperatures chosen to promotethe steps involved in the particular nucleic acid amplification (e.g.,annealing of primer to nucleic acid target, primer extension anddissociation of strands in amplified product). In some instances, it isadvantageous to separately control temperature between different columns(e.g., because different primers are introduced into different columns).To separately regulate the temperature, each column of themicrofabricated device shown in FIG. 5 may include an associatedtemperature control structure. Although not shown in FIG. 5, suchtemperature control structures are described in detail below in sectionVIII and include thermistors, resistive heaters and Peltier controllers.

In other instances, separate control over the temperatures of columns ofthe array shown in FIG. 5 is not required. Such may be the case when thesame primer is introduced to various samples in each column or when theprimers and target nucleic acids have sufficiently similar compositionthat the amplification reaction steps can be performed at the sametemperatures. Various temperature control devices can be utilized inthis situation, including those described in section VIII. One option issimply to position the device adjacent a temperature control block whichprovides uniform heat over the array surface and which can be cycledthrough the different temperatures required for amplification.

If a very high degree of temperature control is required (e.g., forstudies conducted to optimize reaction conditions), the devices can bemanufactured such that the temperature at each junction can beseparately regulated. Resistive heaters (see infra) positioned at eachjunction is one option for forming such devices, although other optionsdescribed in section VIII can be utilized as well.

2. Methods for Conducting Analyses

FIGS. 6A-6C show enlarged plan views of adjacent junctions of array 500of FIG. 5. For purposes of illustration, the control lines are omittedin FIGS. 6A-6C. In addition, the lateral distance between junctions isconsiderably shortened, and in actuality the junctions would beseparated by a substantial distance to prevent cross-contaminationbetween junctions.

In a first step shown in FIG. 6A, column valves 516 are closed andnucleic acid samples are first flowed down each of horizontal flowchannels 504. In the array portion shown enlarged in FIG. 6A, inter-rowvalve regions 526 are thereby charged with sample material S1.

Next, as shown in FIG. 6B, row valves 518 are closed, and column valves516 are opened. Solutions containing different primers are flowed downeach of vertical flow channels 506. In the array portion enlarged inFIG. 6B, junctions 502 are thereby charged with primers P1 and P2.

Finally, as shown in FIG. 6C, column valves 516 are closed and rowvalves 518 are opened. Pumping of the peripheral peristaltic pumps ofthe array causes the nucleic acid sample in inter-valve regions 526 tomingle with the primer solution of junctions 502 as both are flowed intojunctions 502 and inter-valve regions 526. In the array portion enlargedin FIG. 6C, amplification may then occur in sample/primer environmentsS1P1 and S1P2 provided the primer and a nucleic acid within a givenjunction are complementary to one another.

With certain other devices, separate control lines can be used tocontrol alternate row valves. In such an embodiment, the inter-row valveregions and the junctions are charged with sample and primer asdescribed above in FIGS. 6A and 6B. Next, alternate row valves areopened such that sample in inter-row valve regions mixes by diffusionwith primer in the junctions. This particular approach does not requirepumping, and the closed state of the other set of alternate row valvesprevents cross-contamination.

Once the various nucleic acid and primer solutions have been introducedinto the array of junctions in the high throughput structure and mixedwith one another (see FIG. 5), the device is cycled through the varioustemperatures which promote the different amplification stages. For thosejunctions in which the primer is complementary to a nucleic acidcontained in the sample, amplified product is formed and cansubsequently be detected. With knowledge of the identity of the primerat each junction, the presence or absence of particular target nucleicacids at each junction can be determined. Given the large number ofjunctions that can be formed as part of a microfluidic device, one caninterrogate a very large number of samples in a short time period.

Amplified products can be detected according to any of the methodsdescribed below under section IX. As described in greater detail infra,one such approach utilizes molecular beacons (see, e.g., Tyagi et al.,(1996) “Molecular Beacons: Probes that Fluoresce upon Hybridization”,Nature Biotechnology 14:303-308, which is hereby incorporated byreference for all purposes). By utilizing a variety of differentmolecular beacons (optionally bearing different labels), one can readilydetect the presence of specific nucleic acids that are complementary tothe target nucleic acid of interest.

Typically, amplified product is detected within the junctions. Incertain designs, each junction is associated with its own detector. Withother designs, each junction is interrogated with a single detector. Forexample, a detector may include a translatable stage to properlyposition each junction with respect to the detection element or ascanner that illuminates each junction. Alternatively, array detectorscan be utilized to detect signals from each of the junctionssimultaneously. Further details regarding these and other options areprovided in Section IX.

B. Second Exemplary Configuration

1. Architecture

Other devices are array-based devices and share a number of similaritieswith the architecture to the arrays just described and illustrated inFIGS. 5 and 6A-6C. In general these devices also comprise a plurality ofintersecting vertical and horizontal flow channels which form an N×Narray of cross-injection junctions or reaction chambers. A separateamplification reaction can be performed at each junction, therebyallowing a very large number of amplification reactions to be carriedout on a single device at the same time. Certain of these devicesinclude two types of valves, a “large valve” and a “small valve,” thatdiffer with respect to the magnitude of the actuation force required toactivate the valve. Thus, these valves have different activationthresholds. Utilization of such valves provides another level of controlin regulating solution flow through the array.

Some devices are also designed such that a plurality of the verticaland/or horizontal flow channels are fluidly connected to a shared input.This configuration is useful because it means that separate aliquots ofsample or reagent need not be separately added to each of the flowchannels. The ability to fill a plurality of flow channels with a singlesample significantly reduces total sample or reactant volume byeliminating much of the dead volume associated with separate injectionsinto each flow channel. This feature is particularly important when theamount of sample or reagent available is limited or the cost of thesample and/or reagent is high.

FIG. 12 illustrates a specific example of such an alternative structurefor performing nucleic acid amplifications, including several of theforegoing design elements. The fluid layer (i.e., the layer containingthe fluid channels) of the particular array-based device shown in ofFIG. 12 comprises an 8×8 array 1200 of cross-injection junctions formedby the intersection of parallel horizontal flow channels and parallelvertical flow channels. The horizontal flow channels 1202 each haveindependent inlets 1204 and outlets 1206. The vertical flow channels, incontrast, are divided into a first set of M vertical flow channels 1208a that are connected to independent inlets 1210 and outlets 1212, and asecond set of M vertical flow channels 1208 b that are connected to asingle or shared inlet 1214 and outlet 1216. The junctions, then, fallinto two groups, those junctions 1218 a formed by the intersectionbetween a horizontal flow channel 1202 and a vertical flow channel 1208a not connected to shared inlet 1214, and those junctions 1218 b thatare formed by the intersection between a horizontal flow channel 1202and a vertical flow channel 1208 b that is connected to the shared inlet1214.

This design enables N sets of a first reagent to be mixed with M sets ofa second reagent and M sets of a third reagent (where M+M=N). Thus, forinstance, with array 1200, four nucleic acid samples (e.g., S1-S4) canbe mixed with eight primers, P1-P8, thus generating a total of 4×8=32simultaneous reactions on a single chip. This particular architecture isuseful because a reagent that is required for each reaction can beintroduced as a single aliquot via shared inlet 1214 into the M verticalflow channels 1208 b that are connected to shared inlet 1214. Thisfeature reduces the complexity in loading the array 1200 and minimizesthe amount of solution required for injection. Other componentsnecessary for conducting a particular amplification reaction (e.g.,labeled and/or unlabeled nucleotides, metal ions and other cofactors)can be included in either the nucleic acid sample and/or with the primersolution.

In the case of nucleic acid amplification reactions, for example,nucleic acid samples S1-S4 and common reagent, R (e.g., polymerase), aretransported along vertical flow channels 1208 a and 1208 b under theaction of peristaltic pumps 1222 a and 1222 b formed by overlyingcontrol channels 1220 a and 1220 b, respectively. Movement of primersolutions P1-P5 along horizontal flow channels 1202 is controlled byperistaltic pumps 1224 a and 1224 b formed by overlying control channels1246 a and 1246 b. respectively. Because peristaltic pumps 1222 a, 1222b, 1224 a and 1224 b are positioned on each side of array 1220, solutioncan readily be flowed back and forth through the vertical flow channels1208 a and 1208 b and the horizontal flow channels 1202.

The control layer that overlays the fluid layer contains two types ofcontrol channels to define two different types of valves. Each set ofcontrol channels can have a single inlet for actuating the controlchannel. The use of the two different types of valves enables horizontalflow channels 1202 and vertical flow channels 1208 a and 1208 b to beactuated separately. The two valves differ with respect to the width ofthe control channel. Valves with wider control channels (e.g.,approximately 200 microns) are referred to as big valves, whereas valveswith narrower control channels (e.g., approximately 75 microns) arereferred to as small valves. The wider the flow channel is, the easierit is to close the flow channel. Thus, at high actuation levels (e.g.,high pressure levels), both big and small valves close and preventsolution flow through the flow channel upon which they act. However, atintermediate actuation levels (e.g., intermediate pressure levels), bigvalves remain closed and block solution flow, whereas small valves aredeactuated and allow solution to flow through the flow channel. At lowactuation levels (e.g., low pressure levels), both big valves and smallvalves are deactuated and allow solution to flow.

With continued reference to FIG. 12, large column valves 1248 that blocksolution flow in the vertical flow channels 1208 a and 1208 b arecontrolled by a single control line 1250. In contrast, another controlline 1252, can regulate solution flow through the horizontal flowchannels 1202 by actuating both large row valves 1254 and small rowvalves 1256. For purposes of illustration, only the first portion ofcontrol lines 1250 and 1252 are shown in FIG. 12. However, it is to beunderstood that every row valve 1254 and 1256 and column valve 1248 iscontrolled by control lines 1250 and 1252. As described in greaterdetail infra, the ability to selectively actuate the large valves andsmall valves means that solution can be flowed through a flow channeland then trapped between a pair of small valves. These small valves canthen be selectively opened while the large valves remain closed to allowdiffusive mixing between the trapped solution and solution within ajunction, for example.

Thus, during an amplification reaction for instance, row valves 1254 and1256 can be opened while column valves 1248 are closed to introduceeight different primer solutions (P1-P8) into horizontal flow channels1202 and then into junctions 1218 a and 1218 b. Next, row valves 1254and 1256 are closed and column valves 1248 opened to allow samplescontaining nucleic acids to flow through the vertical flow channels 1208a (i.e., those that are connected to individual inlets 1210) intojunctions 1218 a. With the valves in this same configuration, polymeraseis introduced into those vertical flow channels 1208 b that areconnected to the shared inlet 1214. Through the metering techniquesdescribed below in connection with FIGS. 13A-D and 14A-C, all 8×4=32possible combinations of nucleic acid sample and primer are stored atthe junctions 1208 a of array 1200.

2. Methods for Conducting Analyses

FIGS. 13A-13D show enlarged plan views of adjacent junctions of array1200 of FIG. 12. To increase clarity, the control lines are omitted inFIGS. 13A-13D. Initially, column valves 1248 are closed and primersolutions are first flowed down each of horizontal flow channels 1202.In the array portion shown enlarged in FIG. 13A, junctions 1218 a and1218 b, and inter-row valve regions 1260 a defined by two large valves1254 and inter-row valve regions 1260 b defined by two small valves 1256are thereby charged with primer, P1.

Next, as shown in FIG. 13B, both large row valves 1254 and small rowvalves 1256 are closed, while column valves 1248 are opened. Nucleicacid samples are then transported down vertical flow channels 1208 a notconnected to shared inlet 1214. In the array portion enlarged in FIG.13B, junctions 1218 a are thereby charged with sample 51. Similarly,polymerase, Pol, is flowed down vertical flow channels 1208 b that areconnected to shared inlet 1214; the junctions 1218 b shown in arrayportion enlarged in FIG. 13B thus become charged with polymerase.Consequently, vertical flow channels alternate between those thatcontain sample and those that contain polymerase.

Finally, as shown in FIG. 13C, column valves 1248 are closed and smallrow valves 1256 are opened, while large row valves 1254 are kept closed.As indicated above, this can be achieved by applying an intermediatepressure to the control channels associated with large and small valves1254 and 1256. In this approach, no pumping along the horizontal flowchannel 1202 is required, as primer P1, sample S1 and polymerase Pol mixby diffusion to form the mixture P1, S1, Pol. Alternatively, as shown inFIG. 13D, column valves 1248 can be closed and both large row valves1254 and small row valves 1256 opened. Pumping of the peripheralperistaltic pumps of the array causes the primer, P1, in inter-valveregions 1260 a to mix with the nucleic acid sample, S1, of junction 1218a and polymerase Pol within junction 1218 b, either as they are pumpedin one direction or as they are pumped back and forth.

The foregoing process is illustrated in greater detail in FIGS. 14A-14C.FIG. 14A shows an enlarged view of a portion of one flow channel 1204.FIG. 14B presents a cross-sectional view along line B-B′ of the enlargedflow channel portion of FIG. 14A prior to deactuation of small rowvalves to allow diffusion of sample, primer and polymerase located ininter-valve regions and adjacent junctions. FIG. 14C shows across-sectional view along line B-B′ of the enlarged flow channelportion of FIG. 14A after deactuation of the small row valves.

Control line 1252 comprises parallel branches 1252 a and 1252 bpositioned on either side of flow channel 1204. Branches 1252 a and 1252b are connected by pairs of wide cross-over portions 1270 and narrowcross over portions 1272, which define large valves 1254 and smallvalves 1256, respectively. As set forth in FIGS. 13A-13D, junctions 1218a and 1218 b are charged with a nucleic acid and polymerase,respectively; intervalve region 1260 a and 1260 b are initially filledwith primer, P1.

As discussed briefly above, the differing width of cross-over portions1270 and 1272 cause elastomer membranes 1274 and 1276 of large valves1254 and small valves 1256, respectively, to have different actuationthresholds. In particular, because wide membranes 1274 have increasedarea, they are easier to actuate then narrow membranes 1276. Thus,application of a high pressure to control line 1252 causes thedeflection of both elastomer membranes 1274 and 1276 into the underlyingflow channel 1204, closing both large valves 1254 and small valves 1256.Application of a low pressure to control line 1252, however, causes bothelastomer membranes 1274 and 1276 to retract out from the underlyingflow channel 1204, thus opening both large valves 1254 and small valves1256. Finally, application of an intermediate pressure to control line1252 causes only the wide membrane 1274 of large valve structures 1254to remain actuated, whereas narrow membranes 1276 of small valvestructures 1256 are deactuated to open the valve.

The differential response of the different sized valves enables aplurality of reaction subchambers to be defined with a single controlchannel. These subchambers can then be opened, permitting diffusion ofthe various reagents to mix together. This is illustrated in FIG. 14C,wherein an intermediate pressure is applied to control line 1252,allowing narrower membranes 1276 of small valve structures 1256 toretract out of flow channel 1204, while wide membranes 1274 of largevalve structures 1254 remain within flow channel 1204 to prevent crosscontamination between reaction sites.

Once the various nucleic acid and primer solutions have been introducedinto the array of junctions in the high throughput structure and mixedwith one another (see FIG. 5), the device is cycled through the varioustemperatures which promote the different amplification stages. For thosejunctions in which the primer is complementary to a nucleic acidcontained in the sample, amplified product is formed and cansubsequently be detected. Any of the variety of detection methodsdisclosed in section IX can be utilized to detect amplified product.

As with the other array-based system described supra, amplified productis usually detected within the junctions. In certain designs, eachjunction is associated with its own detector. With other designs, eachjunction is interrogated with a single detector (e.g., via the use of atranslatable stage or an array detector).

Thus, the foregoing matrix arrays provides a way to conduct a largenumber of analyses with limited sample volume. Instead of having tomicropipette an aliquot of sample (typically about 1 microliter) onto amicrofluidic device for each analysis, the array- or matrix-baseddevices provided herein enable a large number of reactions to beconducted with a single aliquot of sample, as the sample (e.g., nucleicacid sample) comes into contact with numerous different reactants (e.g.,different primers) in the various junctions which it enters. Thus, forinstance, a one microliter sample can provide 500 assays (assuming 2nanoliters per assay) using an array-based device such as set forthherein.

Further discussion of matrix arrays such as those disclosed herein thatcan be utilized in similar types of analyses, as well as additionaldiscussion on the structure of such devices is set forth in a commonlyowned and copending application entitled “High Throughput Screening ofCrystallization of Materials,” filed Apr. 5, 2002, and having attorneydocket number 20174C-0049200US.

VII. Fabrication of Microfluidic Devices

The microfluidic devices disclosed herein are typically constructed bysingle and multilayer soft lithography (MLSL) techniques and/orsacrificial-layer encapsulation methods. The MLSL approach involvescasting a series of elastomeric layers on a micro-machined mold,removing the layers from the mold and then fusing the layers together.In the sacrificial-layer encapsulation approach, patterns of photoresistare deposited wherever a channel is desired. The use of these techniquesto fabricate elements of microfluidic devices is described, for example,by Unger et al. (2000) Science 288:113-116, by Chou, et al. (2000)“Integrated Elastomer Fluidic Lab-on-a-chip-Surface Patterning and DNADiagnostics, in Proceedings of the Solid State Actuator and SensorWorkshop, Hilton Head, S.C.; and in PCT Publication WO 01/01025, all ofwhich are incorporated herein by reference in their entirety for allpurposes.

More specifically, certain fabrication methods involve initiallyfabricating mother molds for top layers (elastomeric layer with thecontrol channels) and bottom layers (elastomeric layer with the flowchannel) on silicon wafers by photolithography with photoresist (ShipleySJR 5740). Channel heights can be controlled precisely by the spincoating rate. Photoresist channels are formed by exposing thephotoresist to UV light followed by development. Heat reflow process andprotection treatment is performed as described previously (M. A. Unger,H.-P. Chou, T. Throsen, A. Scherer and S. R. Quake, Science 288, 113(2000)). Thereafter, a mixed two-part-silicone elastomer (GE RTV 615) isspun into the bottom mold and poured onto the top mold, respectively.Again, spin coating can be utilized to control the thickness of bottompolymeric fluid layer. After baking in the oven at 80° C. for 25minutes, the partially cured top layer is peeled off from its mold,aligned and assembled with the bottom layer. A 1.5-hour final bake at80° C. is used to bind these two layers irreversibly. Once peeled offfrom the bottom silicon mother mold, this RTV device is typicallytreated with HCL (0.1N, 30 min at 80° C.) to cleave some of the Si—O—Sibonds to expose hydroxy groups that make the channels more hydrophilic.The device can the be sealed hermetically to a support. The support canbe manufactured of essentially any material. The surface should be flatto ensure a good seal as the seal formed is primarily due to adhesiveforces. Examples of suitable supports include glass, plastics and thelike. The device can even be directly attached to certain detectors suchas CCD elements and CMOS detection elements.

The devices formed according to the foregoing method results in thesubstrate (e.g., glass slide) forming one wall of the flow channel. Suchan arrangement can be useful as certain temperature control elements caneasily be formed onto the substrate. For example, as described ingreater detail infra in the section on temperature control elements,certain thermistor heaters can be formed by sputtering a resistive metalsuch as tungsten onto the substrate utilizing established techniques.However, in some instances the device once removed from the mother moldis sealed to a thin elastomeric membrane such that the flow channel istotally enclosed in elastomeric material. The resulting elastomericdevice can then optionally be joined to a substrate support. Deviceshaving this latter structure can be useful for analyses are expected togenerate high backpressures. Such pressures can sometimes cause the sealbetween the elastomeric device and the substrate to fail.

It has also been found that the seal between the elastomeric structureand the substrate can be improved by cleaning the elastomeric structurewith ethanol prior to placing the structure on the substrate.

Further details regarding the preparation of a rotary pump such asutilized in some of the devices provided herein is set forth in Example1 infra

VIII. Temperature Control

A number of different options of varying sophistication are availablefor controlling temperature within selected regions of the microfluidicdevice or the entire device. Thus, as used herein, the term temperaturecontroller is meant broadly to refer to a device or element that canregulate temperature of the entire microfluidic device or within aportion of the microfluidic device (e.g., within a particulartemperature region or at one or more junctions in an array or matrixmicrofluidic device). With the devices and methods described herein,typically the temperature controllers are used to maintain a particulartemperature within each of the temperature regions. Although in certainapplications it may be advantageous to adjust the temperature within oneor more of the temperature regions.

A. Resistive Heaters

A resistive heater is an example of one heating element that can beutilized. Such heating elements can be adjusted to maintain a particulartemperature. FIG. 4 illustrates the use of such a resistive heater. FIG.4 is a cross-sectional view of a region of a microfluidic device formedof a control layer 402 into which a control channel 404 is formed. Thiscontrol channel 404 overlays a flow channel 406 formed in fluid layer408 which itself is attached to a substrate 410. One method forproducing a heating element 412 of this type is to sputter a metal(e.g., tungsten) on the support 410 (e.g., glass slide) to which thefluid layer elastomeric structure 408 is applied. The sputtered material(i.e., heating element 412) is typically applied as a very thin layer(e.g., 500 to 1000 Angstroms thick) on support 410. To facilitatealignment of a section of the flow channel 406 with the heating element412, the heating element 412 is often made wider than the flow channel406. Because the heating element 412 is so thin, the elastomericstructure 408 having the flow channel 406 molded therein can form atight seal against the heating element 412.

Typically, different resistive heaters are placed at each of thedifferent temperature regions and regulated by their own power supply.However, the elastomeric devices can also be placed on a single heaterthat is regulated by a single power supply. The temperature at any giventemperature region can then be controlled by the thickness of the metalsputtered adjacent each temperature region.

B. Peltier Heaters

Another suitable temperature controller is a Peltier controller (e.g.,INB Products thermoelectric module model INB-2-(11-4)-1.5). Thiscontroller is a two-stage device capable of heating to 94° C. Such acontroller can be utilized to achieve effective thermal cycling or tomaintain isothermal incubations at any particular temperature.

C. Heat Exchangers

In some devices and applications, heat exchangers can also be utilizedin conjunction with one of the temperature control sources to regulatetemperature. Such heat exchangers typically are made from variousthermally conductive materials (e.g., various metals and ceramicmaterials) and are designed to present a relatively large externalsurface area to the adjacent region. Often this is accomplished byincorporating fins, spines, ribs and other related structures into theheat exchanger. Other suitable structures include coils and sinteredstructures. In certain devices, heat exchangers such as these arepositioned adjacent to the flow channel within one of the temperatureregions or placed within the flow channel or reaction chamber. Heatexchangers that can be utilized with certain devices are discussed, forexample, in U.S. Pat. No. 6,171,850.

FIG. 17 shows a plan view one embodiment of a heat exchanger for use inconjunction with a microfabricated structure in accordance with thepresent invention. Heat exchanger 1700 includes chambers 1702, 1704, and1706 containing a flowable liquid. Liquid in each of chambers 1702,1704, and 1706 is continuously maintained at different temperatures T1,T2, and T3. Serpentine flow channel 1708 is in fluid communication withchambers 1702, 1704, and 1706 through valves 1710, 1712, and 1714respectively.

Serpentine flow channel 1708 is positioned proximate to microfabricatedreaction chamber 1716 having inlet 1716 a and outlet 1716 b,respectively. In the structure shown in FIG. 17, serpentine flow channeloverlies chamber 1716 and is separated from chamber 1716 by a membraneof elastomer material Depending upon actuation of valves 1710, 1712, and1714, the temperature of liquid flowed through serpentine flow channel1708 can be rapidly changed, with the corresponding exchange of thermalenergy occurring across the thin elastomer membrane over the largesurface area defined by serpentine channel 1708.

D. ITO Heaters

Other heating elements are formed from indium tin oxide, which is atransparent conductor. This material is available from a number ofsources including Delta Technologies, of Stillwater, Minn. One methodfor forming an indium tin oxide heating element is to deposit a thinfilm of the material on a substrate (e.g., thin glass slide); this filmis then formed into the desired pattern by etching. Typical etchingsolutions contain 20% HCl and 5% HNO₃. Premixed etching solution isavailable from Cyantek Corp. Alternatively, the material can bepatterned by sputtering and lift off techniques that are known in theart.

ITO heating elements are useful in conducting certain analyses becauseof their transparency. This feature means that reactions (e.g., PCRreactions) can be monitored in real time. This capability is importantbecause it enables quantitative information regarding expression levelsto be determined (see the discussion on quantitative PCR techniquessupra).

D. Chamber Thermocyclers

Certain temperature controllers are designed to cycle the temperature ofan entire microfluidic device such as those disclosed herein. One suchtemperature controller includes a thermoelectric module (e.g., fromOmega) that can be positioned adjacent a microfluidic device. Thisthermoelectric module is typically powered by a direct current powersupply. A programmable temperature controller can be connected to themodule to have the module produce the desired temperature cycle.

Another temperature controller generally consists of a thin chamberformed between two spaced-apart plates, including a top plate and abottom plate, with the spaced-plates connected to at least one inlet andone outlet through which fluids of different temperatures can be flowed.Typically, the chamber includes two inlets positioned at one end of thechamber and an outlet through which fluid in the chamber can exit.Having two inlets means that two fluids at different temperatures can beflowed into the chamber separately and simultaneously, or that one canrapidly switch which of two fluids are introduced into the chamber. Thetop plate of the spaced-apart plates is intentionally made very thin sothat its thermal resistance is negligible compared to that of the bottomplate and the microfluidic chip that rests upon it. As a consequence,there is little difference in temperature between the heat transportfluid and the microfluidic chip, and a negligible heating and coolingramp, thus enabling a temperature cycle can to be established withlittle transition time between temperatures. The can also beappropriately thickness of the temperature controller is sized such thatit can be utilized upon a standard microscope stage or similar devicewhich has a narrow field of view, typically less than 5 mm, as well asbeing at least partially manufactured of optically transparent materialsso that reactions within the microfluidic device can be monitored bymicroscopy or by other visual or spectroscopic means. Thus, the deviceis designed to permit simultaneous optical access in combination withthe ability to both heat and cool the microfluidic device.

Two examples of such a device are illustrated in FIGS. 15A-15D. Thefirst exemplary device is shown in FIGS. 15A and 15B. This particulartemperature controller 1500 includes a bottom glass plate 1502 and anextremely thin (compared to the rest of the device) top glass plate 1501of negligible thermal resistance that are spaced apart by a gasket 1503.The gasket 1503 is shaped in the form of a hollow loop and is sized suchthat it extends around the outer peripheral edge of the top plate 1501.Thus, the space between the bottom plate 1502 and the top plate 1501 asfurther defined by the peripheral gasket 1503 defines a water-tightchamber 1515.

Connector blocks 1506 a and 1506 b are affixed to each end of the topplate 1501. The connector blocks 1506 a and 1506 b each have a cavity1522 a and 1522 b, respectively, that extends into the central region ofone face of the connector block, without extending the entire length ofthe connector block. The cavity of each connector block 1506 a and 1506b is positioned such that it overlays one or more holes 1505 in eitherend of the top plate 1501. This arrangement means that the cavity 1522 aand 1522 b of each connector block is in fluid communication with thechamber 1515 formed between the top plate 1501 and bottom plate 1502 viathe hole(s) 1505 in the top plate. The two connector blocks 1506 a and1506 b and the portion of the upper surface of the top plate 1501extending therebetween define a bed 1514 onto which a microfluidic chip1516 can be placed.

A first and second inlet 1504 a and 1504 b are inserted into opposingends of first connector block 1506 a and each is in fluid communicationwith the cavity 1522 a of first connector block 1506 a. Consequently,fluid introduced via first and second inlets 1504 a and 1504 b can flowinto the cavity 1522 a of first connector block 1506 a and subsequentlypass through the hole(s) 1505 in the top plate 1501 and into the chamber1515. Similarly, an outlet 1504 c is positioned in one end of the secondconnector block 1506 b. Outlet 1504 c is in fluid communication with thecavity 1522 b of connector block 1506 b, which means that fluid in thechamber 1515 can flow up through the hole(s) 1505 in the top plate 1501into the cavity 1522 b of second connector block 1506 b where the fluidcan then exit via outlet 1504 c.

Inlets 1504 a and 1504 b are each connected to a temperature bath (notshown) by a connecting line (not shown). The temperature of the fluid ineach bath is set so fluid delivered to the temperature controller willbe one of the temperatures in the desired temperature cycle. Eachtemperature bath is further connected to a pump (not shown) to pumpfluid from the temperature bath into the chamber of the temperaturecontroller. Typically, the connecting lines are protected to guardagainst heat loss. One option is to include the connecting line in aflowing fixed temperature water jacket. Because the top plate is verythin, heat can be rapidly transferred to or from the microfluidic chippositioned on its upper surface. Generally the top plate 1501 is lessthan 1 millimeter thick, and in other instances less 500 microns, instill other instances less than 250 microns, and in still otherinstances less than 100 microns.

In operation, microfluidic chip 1516 is placed into bed 1514 such thatit rests against the upper face of the top plate 1501. The temperatureof the temperature baths are set to the temperatures appropriate for thetemperature cycle for the analysis or reaction. In the case of nucleicacid amplification reactions, the temperatures are selected to promotethe different stages of the amplification process. By way ofillustration but not limitation, one water bath is heated to 97° C., andthe other to 60° C. Fluid from the bath at the initial temperature ofthe cycle is flowed into chamber 1515 via first inlet 1504 a and exitsvia outlet 1504 c. The thinness of top plate 1501 and resulting lowthermal resistance permits the substrate 1520 of microfluidic chip 1516to be heated and cooled very rapidly. When the temperature is to bechanged during a temperature cycle, fluid from the second temperaturebath at the next temperature in the cycle is flowed into second inlet1504 b, and flow of fluid from the first temperature bath is stopped. Asfluid at the second temperature is flowed into chamber 1515, fluidcontinues to flow out of chamber 1515 via outlet 1504 c.

Another temperature controller 1550 is shown in FIGS. 15C and 15D andhas a design similar to the device illustrated in FIGS. 15A and 15B. Theprimary difference is with respect to the construction of the top plate1507. In the thermocycler shown in FIGS. 15C and 15D, the top plate 1507is part of an assembly 1525 in which the top plate 1507 is connected bya hinge 1511 to a sealing plate 1510. For increased durability, the topplate 1507 and the sealing plate 1510 are made of metal. Because thethermocycler is preferably transparent to facilitate optical detection,both the top plate 1507 and the sealing plate 1510 contain centrallylocated holes 1513 and 1512, respectively (the central hole 1513 in topplate 1507 is in addition to the hole(s) 1505 located at either end).Central holes 1512 and 1513 are aligned such that when the sealing plate1510 is folded down onto top plate 1507, the central holes 1512 and 1513align with one another. The central hole 1513 in the top plate 1507 issized such that it is smaller than the substrate 1520 upon which theelastomeric structure 1518 of microfluidic chip 1516 is attached. Thus,when microfluidic chip 1516 is placed in the bed 1514 defined by theupper face of the top plate 1507 and the two connector blocks 1506 a and1506 b, the substrate 1520 spans the central hole 1513 of the top plate1507. The central hole 1512 in the sealing plate 1510 is also positionedsuch that when microfluidic chip 1516 is placed in the bed 1514, thecentral hole 1512 fits around the elastomeric structure 1518, with apart of the elastomeric structure 1518 extending through the centralhole 1512 of the sealing plate 1510. Latch 1509 b is closed upon catch1509 a, thereby forcing the microfluidic chip 1516 against top plate1507.

In order to prevent fluid from flowing through the central hole 1513 inthe top plate 1507 and out past the edges of the microfluidic chip 1516when it is positioned in bed 1514, two additional gaskets 1530 and 1532are typically utilized to form a water-tight seal. A first gasket 1530is attached to either the upper face of the top plate 1507 around theperiphery of the central top plate hole 1513, or placed on the undersideof the substrate 1520 of the microfluidic chip 1516 itself. A secondgasket 1532, is either attached to the under surface (assuming the plateis closed) of the sealing plate 1510 and runs around the periphery ofthe central sealing plate hole 1512, or is simply attached to the uppersurface of the substrate 1520 of the microfluidic chip 1516.

Thus, in operation, the sealing plate 1510 is lifted up away from thetop plate 1507 so that microfluidic chip 1516 can be placed onto bed1514, with the elastomeric structure 1518 of the microfluidic chip 1516being disposed over the central hole 1513 in the top plate 1507. Thesealing plate 1510 is then closed such that the underside of the sealingplate 1510 is adjacent the upper surface of the top plate 1507, with thesubstrate 1520 of the microfluidic chip 1516 being sandwichedtherebetween, and the elastomeric structure 1518 extending up throughthe central hole 1512 in the sealing plate 1510. Fluids of differingtemperature can then be cycled through the chamber 1515 as describedwith respect to the temperature controller 1500 shown in FIGS. 15A and15B via inlets and outlet.

Because the elastomeric structure 1518 is coincident with the centraltop plate hole 1513 and the central sealing plate hole 1512, reactionswithin the microfluidic chip 1516 can be monitored by microscopy orother optical methods. Further, as alluded to above, this particulardesign permits the thermocycling device to be made out of sturdiermaterials (e.g., metal), and is thus reusable and has a longer life thanthat expected for other devices such as that depicted in FIGS. 15A-B.

While the temperature controller has been described as being formed ofglass or metal plates, the plates can be formed of any of a number ofmaterials, including, but not limited to, plastics, ceramics andcomposite materials. However, the use of transparent materials such asglass plates (e.g., microscope coverslips) permit optical access toreactions within the microfluidic device while simultaneously providingthe required temperatures, thus allowing detection of reaction productsand monitoring of the reactions in real time.

Likewise, the gaskets can be formed of a number of different materialsto provide an adequate seal between the bottom and top plates. Examplesof suitable gasket materials or sealants include a wide variety ofadhesives, such as PDMS (Polydimethly Siloxane), silicon, or appropriateepoxies.

A number of different fluids can be utilized with this type oftemperature controller, provided the fluid has a sufficiently lowviscosity such that it readily flows through the chamber and preferablydoes not form bubbles that would disrupt optical viewing. Examples ofsuitable fluids include a solution of water and Ethylene Glycol in equalproportions and heat transfer oil.

E. Temperature Sensors

As described above, performance of thermal cycling steps involvingheating/cooling of reactants is an important part of the process ofamplification of nucleic acids. In order to ensure the accuracy of thesethermal cycling steps, in certain devices it is therefore useful toincorporate sensors detecting temperature in the various temperatureregions.

One structure for detecting temperature in accordance with the presentinvention is a thermocouple. Such a thermocouple could be created asthin film wires patterned on the underlying substrate material, or aswires incorporated directly into the microfabricated elastomer materialitself.

Temperature may also be sensed through a change in electricalresistance. For example, change in resistance of a thermistor fabricatedon an underlying semiconductor substrate utilizing conventionaltechniques could be calibrated to a given temperature change.

Alternatively, a thermistor could be inserted directly into themicrofabricated elastomer material. Still another approach to detectionof temperature by resistance is described in Wu et al. in “MEMS FlowSensors for Nano-fluidic Applications”, Sensors and Actuators A 89152-158 (2001), which is hereby incorporated by reference in itsentirety. This paper describes the use of doped polysilicon structuresto both control and sense temperature. For polysilicon and othersemiconductor materials, the temperature coefficient of resistance canbe precisely controlled by the identity and amount of dopant, therebyoptimizing performance of the sensor for a given application.

Thermo-chromatic materials are another type of structure available todetect temperature on regions of an amplification device. Specifically,certain materials dramatically and reproducibly change color as theypass through different temperatures. Such a material could be added tothe solution as they pass through different temperatures.Thermo-chromatic materials could be formed on the underlying substrateor incorporated within the elastomer material. Alternatively,thermo-chromatic materials could be added to the sample solution in theform of particles.

Another approach to detecting temperature is through the use of aninfrared camera. An infrared camera in conjunction with a microscopecould be utilized to determine the temperature profile of the entireamplification structure. Permeability of the elastomer material toradiation would facilitate this analysis.

Yet another approach to temperature detection is through the use ofpyroelectric sensors. Specifically, some crystalline materials,particularly those materials also exhibiting piezoelectric behavior,exhibit the pyroelectric effect. This effect describes the phenomena bywhich the polarization of the material's crystal lattice, and hence thevoltage across the material, is highly dependent upon temperature. Suchmaterials could be incorporated onto the substrate or elastomer andutilized to detect temperature.

Other electrical phenomena, such as capacitance and inductance, may beexploited to detect temperature in accordance with embodiments of thepresent invention.

IX. Detection

A. General

The microfluidic devices provided can be utilized in combination with awide variety of detection methodologies. The particular detection systemutilized depends upon the particular type of event and/or agent beingdetected. Examples of particular detection methods useful with thepresent microfluidic devices include, but are not limited to, lightscattering, multichannel fluorescence detection, UV and visiblewavelength absorption, luminescence, differential reflectivity, andconfocal laser scanning Applications can also utilize scintillationproximity assay techniques, radiochemical detection, fluorescencepolarization, fluorescence correlation spectroscopy (FCS), time-resolvedenergy transfer (TRET), fluorescence resonance energy transfer (FRET)and variations such as bioluminescence resonance energy transfer (BRET).Additional detection options include electrical resistance, resistivity,impedance, and voltage sensing.

The term “detection section,” “detection region,” and other like termsrefer to the portion of the microfluidic device at which detectionoccurs. In general, the detection section can be at essentially anypoint along one of the flow channels or at an intersection of flowchannels. As indicated supra, for the devices shown in FIGS. 1 and 2,the detection section can include the central loop (FIG. 1) or a portionthereof, or one or more reaction chambers (FIG. 2). Alternatively,detection can occur at another region on the device or off the deviceonce solution containing unreacted reagents and products has beenwithdrawn from the flow channel in which thermocycling has occurred.

Similarly, detection with the matrix array devices discussed above andillustrated in FIGS. 5 and 12, for example, typically occurs withinregions of flow channels that are adjacent a junction or within thejuctions themselves.

The detection region can be in communication with one or moremicroscopes, diodes, light stimulating devices (e.g., lasers),photomultiplier tubes, processors and combinations of the foregoing,which cooperate to detect a signal associated with a particular eventand/or agent. Often the signal being detected is an optical signal thatis detected in the detection section by an optical detector. The opticaldetector can include one or more photodiodes (e.g., avalanchephotodiodes), a fiber-optic light guide leading, for example, to aphotomultiplier tube, a microscope, and/or a video camera (e.g., a CCDcamera).

An optical detector can be microfabricated within the microfluidicdevice, or can be a separate element. If the optical detector exists asa separate element and the microfluidic device includes a plurality ofdetection sections, detection can occur within a single detectionsection at any given moment. In other instances, an automated system isutilized which scans the light source relative to the microfluidicdevice, scans the emitted light over a detector, or includes amultichannel detector. For example, the microfluidic device can beattached to a translatable stage and scanned under a microscopeobjective. The acquired signal is routed to a processor for signalinterpretation and processing. Arrays of photomultiplier tubes can alsobe utilized. Additionally, optical systems that have the capability ofcollecting signals from all the different detection sectionssimultaneously while determining the signal from each section can beutilized.

In some instances, the detection section includes a light source forstimulating a reporter that generates a detectable signal. The type oflight source utilized depends in part on the nature of the reporterbeing activated. Suitable light sources include, but are not limited to,lasers, laser diodes and high intensity lamps. If a laser is utilized,the laser can be utilized to scan across a set of detection sections ora single detection section. Laser diodes can be microfabricated into themicrofluidic device itself. Alternatively, laser diodes can befabricated into another device that is placed adjacent to themicrofluidic device being utilized to conduct a thermal cycling reactionsuch that the laser light from the diode is directed into the detectionsection.

In some instances in which external radiation and/or an externaldetector is/are utilized, a substrate that is optically transparent atthe wavelength being monitored is used to cover the detection section.However, by appropriate selection of elastomeric materials, monolithicelastomeric devices can still be utilized in conjunction with a widevariety of external optical detection methods. The present devices canutilize a number of optical detection systems that are not possible withconventional silicon-based microfluidic devices because the provideddevices typically utilize elastomers that are substantially opticallytransparent.

Detection can involve a number of non-optical approaches as well. Forexample, the detector can also include, for example, a temperaturesensor, a conductivity sensor, a potentiometric sensor (e.g., pHelectrode) and/or an amperometric sensor (e.g., to monitor oxidation andreduction reactions).

In certain methods, solutions are transported from the microfluidicdevice to a separate external device for further analysis. The externaldevice can be any of a number of analytical devices such as UV/VIS, IR,NMR and/or ESR spectrometers; chromatographic columns (e.g., HPLC);electrophoretic columns and/or mass spectrometry, for example.

B. Detection of Amplified Nucleic Acids

When the devices provided herein are utilized to conduct nucleic acidamplification reactions, a number of different approaches can beutilized to detect amplified product. Examples of suitable approachesinclude the following:

1. Intercalation Dyes

One method for detecting the formation of amplified product utilizeslabels, such as dyes, that only bind to double stranded DNA. In thistype of approach, amplification product (which is double stranded) bindsdye molecules in solution to form a complex. With the appropriate dyes,it is possible to distinguish between dye molecules free in solution anddye molecules bound to amplification product. For example, certain dyesfluoresce only when bound to amplification product. Examples of suitabledyes include, but are not limited to, SYBR™ and Pico Green (fromMolecular Probes, Inc. of Eugene, Oreg.), ethidium bromide, propidiumiodide, chromomycin, acridine orange, Hoechst 33258, Toto-1, Yoyo-1, andDAPI (4′,6-diamidino-2-phenylindole hydrochloride). Additionaldiscussion regarding the use of intercalation dyes is provided by Zhu etal., Anal. Chem. 66:1941-1948 (1994), which is incorporated by referencein its entirety.

2. Size Separation

In this approach, amplified product can be separated from primers andother unreacted reagents and subsequently detected by utilizing varioussize-separation techniques. Amplified product can be readilydistinguished from any unextended primer because of the significantlylarger size of the amplified product. Typically, separation is achievedby withdrawing solution that has completed the thermal cycling processand that includes amplified product and introducing the withdrawn sampleinto a matrix capable of separating nucleic acids on the basis of size.

Some of the microfluidic devices that are provided herein incorporate aseparation module that is in fluid communication with the flow channelin which thermal cycling occurred (e.g., the central flow loop or flowchannel with reaction chambers; see FIGS. 1 and 2). In such modules,various size exclusion materials are packed. Typically, the sizeexclusion material is an electrophoretic gel matrix. Thus, for example,separation can be achieved by capillary gel electrophoresis. Of course,the size separation process can also be conducted off the microfluidicdevice. Thus, for instance, sample removed after thermal cycling can beseparated on a dedicated capillary gel electrophoresis apparatus or viahigh pressure liquid chromatography (HPLC). Regardless of whetherseparation is achieved on the microfluidic device or off, amplifiedproduct can be detected by staining the gel with intercalating dyes (seesupra) or by using labeled primers, for example.

In yet another option, the microfluidic device either includes or is influid communication with a microfluidic DNA sizer that employs certainof the same elastomeric elements of the present devices. These DNAsizers enable one to selectively separate different nucleic acidmolecules according to size. Such sizers are discussed, for example, byChou, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:11-13, which isincorporated herein by reference in its entirety for all purposes.

3. Probe-Based Detection Methods

These detection methods involve some alteration to the structure orconformation of a probe caused by hybridization of the probe to thetarget nucleic acid, primer extension or some other event. Specificexamples of such approaches follow:

Quantitative RT-PCR. A variety of so-called “real time amplification”methods or “real time quantitative PCR” methods can also be utilized todetermine the quantity of a target nucleic acid present in a sample bymeasuring the amount of amplification product formed during theamplification process itself. Fluorogenic nuclease assays are onespecific example of a real time quantitation method which can be usedsuccessfully with the methods described herein. The basis for thismethod of monitoring the formation of amplification product is tomeasure continuously PCR product accumulation using a dual-labeledfluorogenic oligonucleotide probe—an approach frequently referred to inthe literature as the “TaqMan” method.

The probe used in such assays is typically a short (ca. 20-25 bases)polynucleotide that is labeled with two different fluorescent dyes. The5′ terminus of the probe is typically attached to a reporter dye and the3′ terminus is attached to a quenching dye, although the dyes can beattached at other locations on the probe as well. The probe is designedto have at least substantial sequence complementarity with the probebinding site on the target nucleic acid. Upstream and downstream PCRprimers that bind to regions that flank the probe binding site are alsoincluded in the reaction mixture.

When the probe is intact, energy transfer between the two fluorophorsoccurs and the quencher quenches emission from the reporter. During theextension phase of PCR, the probe is cleaved by the 5′ nuclease activityof a nucleic acid polymerase such as Taq polymerase, thereby releasingthe reporter from the polynucleotide-quencher and resulting in anincrease of reporter emission intensity which can be measured by anappropriate detector.

One detector which is specifically adapted for measuring fluorescenceemissions such as those created during a fluorogenic assay is the ABI7700 manufactured by Applied Biosystems, Inc. in Foster City, Calif.Computer software provided with the instrument is capable of recordingthe fluorescence intensity of reporter and quencher over the course ofthe amplification. These recorded values can then be used to calculatethe increase in normalized reporter emission intensity on a continuousbasis and ultimately quantify the amount of the mRNA being amplified.

Additional details regarding the theory and operation of fluorogenicmethods for making real time determinations of the concentration ofamplification products are described, for example, in U.S. Pat. No.5,210,015 to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S.Pat. No. 5,863,736 to Haaland, as well as Heid, C. A., et al., GenomeResearch, 6:986-994 (1996); Gibson, U. E. M, et al., Genome Research6:995-1001 (1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA88:7276-7280, (1991); and Livak, K. J., et al., PCR Methods andApplications 357-362 (1995), each of which is incorporated by referencein its entirety.

Some RT-PCR reactions are conducted in a somewhat different fashion. Forexample, amplified product in these reactions is detected by conductingreactions in the presence of dyes that preferentially bind to doublestranded DNA (e.g., SYBR GREEN) and that only generate signal oncebound. Thus, as the amplification reaction progresses, an increasingamount of dye becomes bound and is accompanied by a concomitant increasein signal.

Molecular Beacons: With molecular beacons, a change in conformation ofthe probe as it hybridizes to a complementary region of the amplifiedproduct results in the formation of a detectable signal. The probeitself includes two sections: one section at the 5′ end and the othersection at the 3′ end. These sections flank the section of the probethat anneals to the probe binding site and are complementary to oneanother. One end section is typically attached to a reporter dye and theother end section is usually attached to a quencher dye.

In solution, the two end sections can hybridize with each other to forma hairpin loop. In this conformation, the reporter and quencher dye arein sufficiently close proximity that fluorescence from the reporter dyeis effectively quenched by the quencher dye. Hybridized probe, incontrast, results in a linearized conformation in which the extent ofquenching is decreased. Thus, by monitoring emission changes for the twodyes, it is possible to indirectly monitor the formation ofamplification product. Probes of this type and methods of their use isdescribed further, for example, by Piatek, A. S., et al., Nat.Biotechnol. 16:359-63 (1998); Tyagi, S, and Kramer, F. R., NatureBiotechnology 14:303-308 (1996); and Tyagi, S. et al., Nat. Biotechnol.16:49-53 (1998), each of which is incorporated by reference herein intheir entirety for all purposes.

4. Capacitive DNA Detection

There is a linear relationship between DNA concentration and the changein capacitance that is evoked by the passage of nucleic acids across a1-kHz electric field. This relationship has been found to be speciesindependent. (See, e.g., Sohn, et al. (2000) Proc. Natl. Acad. Sci.U.S.A. 97:10687-10690). Thus, in certain devices, nucleic acids withinthe flow channel (e.g., the substantially circular flow channel of FIG.1 or the reaction chambers of FIG. 2) are subjected to such a field todetermine concentration of amplified product. Alternatively, solutioncontaining amplified product is withdrawn and then subjected to theelectric field.

5. Incorporation of Labeled Primers and/or Nucleotides

In certain reactions, labeled primers and/or nucleotides are utilized.Hence, product formed as the result of primer extension is labeledbecause of the labeled primer and/or the labeled nucleotides that areincorporated into the extension products. A wide variety of labels canbe utilized to label the primer and/or nucleotides. Examples of suitablelabels are listed supra in the definition section.

X. Variations

A. Types of Amplification Reactions

Although some of the foregoing discussion has focused on the use of thepresent microfluidic devices to conduct PCR reactions, the devices canbe utilized to conduct essentially any type of amplification reaction,especially those that involve thermal cycling. The amplificationreactions can be linear amplifications, (amplifications with a singleprimer), as well as exponential amplifications (i.e., amplificationsconducted with a forward and reverse primer set).

Examples of the types of amplification reactions that can be conductedwith the microfluidic devices disclosed herein include, but are notlimited to, (i) polymerase chain (see generally, PCR Technology:Principles and Applications for DNA Amplification (H. A. Erlich, Ed.)Freeman Press, NY, N.Y. (1992); PCR Protocols: A Guide to Methods andApplications (Innis, et al., Eds.) Academic Press, San Diego, Calif.(1990); Mattila et al., Nucleic Acids Res. 19: 4967 (1991); Eckert etal., PCR Methods and Applications 1: 17 (1991); PCR (McPherson et al.Ed.), IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202 and 4,683,195);(ii) ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560(1989) and Landegren et al., Science 241:1077 (1988)); (iii)transcription amplification (see Kwoh et al., Proc. Natl. Acad. Sci. USA86:1173 (1989)); (iv) self-sustained sequence replication (see Guatelliet al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)); and (v) nucleic acidbased sequence amplification (NASBA) (see, Sooknanan, R. and Malek, L.,BioTechnology 13: 563-65 (1995)). Each of the foregoing references areincorporated by reference in their entirety for all purposes.

B. Multiplexing

The microfluidic devices provided herein can be utilized to conductanalyses in a variety of multiplexing format, thus allowing multipleanalyses to be conducted simultaneously. Multiplexing in some formatsinvolves preparing a microfluidic device that incorporates multipledevices of the designs described supra. Such devices are readilyprepared according to the multilayer soft lithographic techniquesdescribed above. Even though a large number of the thermal cyclingdevices can be incorporated into a single microfluidic device, controlof the multiple devices is not rendered unduly complicated. In fact, thedifferent thermal cycling devices can be formed such that solution flowthrough each device can be driven by the same set of control channels.

Multiplex amplifications can even be performed with a single thermalcycling device (e.g., as illustrated in FIGS. 1 and 2). This isaccomplished, for example, by utilizing a plurality of primers, eachspecific for a particular target nucleic acid of interest, during thethermal cycling process. The presence of the different amplifiedproducts can be detected using differentially labeled probes to conducta quantitative RT-PCR reaction or by using differentially labeledmolecular beacons (see supra). In such approaches, each differentiallylabeled probes is designed to hybridize only to a particular amplifiedtarget. By judicious choice of the different labels that are utilized,analyses can be conducted in which the different labels are excitedand/or detected at different wavelengths in a single reaction. Furtherguidance regarding the selection of appropriate fluorescent labels thatare suitable in such approaches include: Fluorescence Spectroscopy(Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al.,Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York,(1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules,2_(nd) ed., Academic Press, New York, (1971); Griffiths, Colour andConstitution of Organic Molecules, Academic Press, New York, (1976);Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland,Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Eugene (1992).

Array type devices such as those described in FIGS. 5 and 12 andaccompanying text can be utilized to perform a very large number ofamplification reactions at once.

C. Immobilized Polymerase

In certain devices, polymerase can be immobilized to a flow channelsurface within the temperature region at which primer extension occurs.In this way, one can avoid the polymerase enzyme becoming denatured inthe high temperature zone. With such an approach, one could then utilizepolymerases from non-thermophile organisms. Such attachment is notpossible with other microfluidic configurations in which the enzyme isused in batch and is heated to high temperature (e.g., 95° C.) in everycycle.

Treatment of the elastomeric device with HCl (0.1N, 30 min 80° C.) makesthe polymer hydrophilic. This treatment breaks some of the Si—O—Si bondsin the silicone polymer used to form the device to create hydroxylgroups that are displayed on the surface of the channel. These hydroxylgroups can be derivatized with appropriate reagents (e.g.3-Aminopropyltriethoxysilane) to introduce amino groups onto thesurface. These amino groups can then be crosslinked with the aminogroups of the protein using various coupling agents (e.g.glutardialdehyde). Alternatively, the polymerase enzyme can also beimmobilized on the glass surface. A wide variety of methods forimmobilizing proteins on different surfaces are described by Gordon, FBickerstaff (Ed), “Immobilization of Enzyme and Cells,” Humana PressInc, 1997.

D. Immobilized Nucleic Acid

In a manner related to immobilization of polymerase within a temperatureregion of one of the microfluidic devices disclosed herein, nucleicacids can also be immobilized within the flow channels of the devices.Usually the nucleic acids are immobilized with a temperature zone orregion at which reaction occurs (see, e.g., FIG. 1), or within areaction chamber or junction (see, e.g., the matrix or array devicesillustrated in FIGS. 5 and 12).

The particular nucleic acid immobilized to the flow channel will dependupon the nature of the analysis being conducted. In the case of nucleicacid amplification reactions, the nucleic acid can be one that is toserve as a template or can be a primer. In other instances, nucleicacids are deposited within a flow channel or array junction to functionas probes that hybridize to complementary target nucleic acids that arepresent within the flow channel or junctions (e.g., nucleic acidextension or amplification products that are formed). Binding between aprobe and target nucleic acid can be detected by a variety of methodsknown in the art. In some instances, the nucleic acid probes aredeposited to form a type of nucleic acid microarray within a flowchannel or junction. By depositing the nucleic acid probes in certainpatterns, detection of the absence or presence of a particular targetnucleic acid can be readily ascertained spatially, i.e, according to thelocation on the microarray at which a signal is generated.

Certain methods for immobilizing nucleic acids parallel the methods justdescribed for immobilizing polymerase. Here, too, elastomeric surfacescan be treated with acid to form hydroxyl groups which can be reacteddirectly with activated nucleic acids or derivatized prior to reaction.Optionally, the activated surface is first reacted with a linker whichis then joined to the desired nucleic acid. Use of a linker can minimizeunwanted steric interferences. As an alternative to attaching thenucleic acid to an elastomeric surface, the nucleic acid can instead beattached to the substrate (e.g., glass) on which the elastomeric layeris placed. In this approach, the nucleic acid is spotted at anappropriate region on the substrate and then the elastomeric layer isoverlayed on the substrate, such that the nucleic acid is positioned inthe desired location of the device.

Further discussion regarding synthesis and deposition of nucleic acidsto form arrays are discussed for example in the following references:Meier-Ewart, et al., Nature 361:375-376 (1993); Nguyen, C. et al.,Genomics 29:207-216 (1995); Zhao, N. et al., Gene, 158:207-213 (1995);Takahashi, N., et al., Gene 164:219-227 (1995); Schena, et al., Science270:467-470 (1995); Southern et al., Nature Genetics Supplement 21:5-9(1999); Cheung, et al., Nature Genetics Supplement 21:15-19 (1999);Beaucage, Tetrahedron Lett., 22:1859-1862 (1981); Needham-VanDevanter,et al., Nucleic Acids Res., 12:6159-6168 (1984); U.S. Pat. Nos.5,143,854, 5,424,186, 5,744,305; PCT patent publication Nos. WO 90/15070and 92/10092; Fodor et al., Science 251:767-777 (1991); and Lipshutz, etal., Nature Genetics Supplement 21:20-24 (1999).

E. Integration with Other Microfluidic Components/Devices

The microfluidic thermal cycling devices provided herein can be utilizedin conjunction with a variety of other microfluidic elements such aspumps, valves, rotary pumps, separation modules and the like,particularly those manufactured from elastomeric materials such as thedevices provided herein (see, e.g., PCT publication WO 01/01025). Thedevices can also be incorporated into microfluidic devices designed toconduct high throughput screening or cell assays, for example. Examplesof such microfluidic devices into which the present devices can beincorporated are described in U.S. provisional application entitled,“Apparatus and Methods for Conducting Cell Assays,” having attorneydocket number 020174-003210US, and filed on even date herewith, and U.S.Provisional application entitled “Apparatus and Methods for ConductingHigh Throughput Screening Assays,” having attorney docket number020174-003220US, and filed on even date herewith, both of which areincorporated by reference in their entirety for all purposes.

F. Combinations of Amplification Reactions

The devices can also be utilized to amplify product amplified during aprevious amplification process (e.g., a prior PCR reaction). Thus, forexample, the devices can be utilized to conduct nested PCR analyses inwhich the PCR product from one amplification reaction acts as templatefor a subsequent PCR utilizing primers that hybridize to segments withinthe amplified product. Reactions of this type are commonly used invarious diagnostic applications.

A second PCR is also needed when performing a RT PCR. Here, an initialRT PCR typically is conducted to generate a full length cDNA from a fulllength transcript. The resulting cDNA library or members thereof cansubsequently be amplified to generate amplification products for use incloning or for generation of hybridization probes.

G. Sample Introduction using a Chip Holder

The inlets to flow channels on certain microfluidic devices are incommunication with on-chip reservoirs or wells. However, in amicrofluidic device requiring the loading of a large number ofsolutions, the use of a corresponding large number of input tubes withseparate interfacing pins may be impractical given the relatively smalldimensions of the fluidic device. In addition, the automated use ofpipettes for dispensing small volumes of liquid is known, and thus ittherefore may prove easiest to utilize such techniques to pipettesolutions directly on to wells present on the face of a chip.

Additionally, capillary action may not be sufficient to draw solutionsfrom on-chip wells into active regions of the chip, particularly wheredead-ended chambers are to be primed with material. In such embodiments,one way of loading materials into the chip is through the use ofexternal pressurization. Again, however, the small dimensions of thedevice coupled with a large number of possible material sources mayrender impractical the application of pressure to individual wellsthrough pins or tubing.

Accordingly, FIG. 16 shows an exploded view of a chip holder 1600designed to hold a chip such as those disclosed herein. Bottom portion1602 of chip holder 1600 includes raised peripheral portion 1604surrounding recessed area 1606 corresponding in size to the dimensionsof chip 1608, allowing microfluidic chip 1608 to be positioned therein.Peripheral region 1604 further defines screw holes 1610.

Microfluidic device 1608 is positioned within recessed area 1606 ofbottom portion 1602 of chip holder 1600. Microfluidic device 1608comprises an active region that is in fluidic communication withperipheral wells 1612 configured in first and second rows 1612 a and1612 b, respectively. Wells 1612 hold sufficient volumes of material toallow device 1608 to function. Wells 1612 may contain, for example,nucleic acid samples, primer solutions or various other reagents.

Top portion 1614 of chip holder 1600 fits over bottom chip holderportion 1602 and microfluidic chip 1608 positioned therein. For ease ofillustration, in FIG. 16 top chip holder portion 1614 is shown invertedrelative to its actual position in the assembly. Top chip holder portion1614 includes screw holes 1616 aligned with screw holes 1610 of lowerholder portion 1602, such that screws 1618 may be inserted through holes1616 to secure the chip 1608 between portions 1614 and 1602 of holder1600.

Lower surface 1614 a of top holder portion 1614 includes raised annularrings 1620 and 1621 surrounding recesses 1622 and 1623, respectively.When top portion 1614 of chip holder 1600 is pressed into contact withchip 1608 utilizing screws 1618, rings 1620 and 1621 press into the softelastomeric material on the upper surface of chip 1608, such that recess1620 defines a first chamber over top row 1612 a of wells 1612, andrecess 1623 defines a second chamber over bottom row 1612 b of wells1612. Holes 1624 and 1626 in the side of top holder portion 1614 are incommunication with recesses 1620 and 1621 respectively, to enable apositive pressure to be applied to the chambers through pins 1628 and1630 inserted into holes 1624 and 1626, respectively. A positivepressure can thus simultaneously be applied to all wells within a row,obviating the need to utilize separate connecting devices to each well.

In operation, solutions are pipetted into the wells 1612, and then chip1608 is placed into bottom portion 1602 of holder 1600. The top holderportion 1614 is placed over chip 11608, and is pressed down by screws.Raised annular rings 1620 and 1621 on the lower surface of top holderportion 1614 make a seal with the upper surface of the chip where thewells are located. Solutions within the wells are exposed to positivepressures within the chamber, and are thereby pushed into the activearea of microfluidic device.

The downward pressure exerted by the chip holder can also preventdelamination of the chip from the substrate during loading. Thisprevention of delamination may enable the use of higher primingpressures.

The chip holder shown in FIG. 16 represents only one possible structure.For example, a chip holder can also include a third portion which fitsover control line outlet ports on the front or back side of the chip,thereby enabling the application of pressure to control lines to controlvalve actuation within the chip. In addition, while the holder describedin FIG. 16 includes a window for viewing of the chip, this may not benecessary if the chip is to be removed from the holder once the chipfilling process is complete.

The chip holder can be equipped with heating elements to provide spatialand temporal temperature profile to the chip positioned therein. Such analternative would eliminate the complexity and expense associated withincorporating heating elements directly onto a substrate that may bedisposable.

In the particular chip holder illustrated in FIG. 16, the top piece ispressed to the chip by turning screws. However, in alternativeembodiments, the downward force could be applied through a press orrobotic arm, thereby potentially eliminating the need for a bottomholder piece.

Furthermore, in the particular chip holder illustrated in FIG. 16, theairtight seal over the wells allowing application of a positive pressureis created by pressing the raised ring into the compliant top surface ofthe elastomer chip. However, a seal could be created by the addition offlexible o-rings to the chip holder. Such o-rings would permit use of achip holder with embodiments of microfluidic devices that feature arigid top surface.

Finally, it is important to recognize that use of a chip holderstructure in accordance with embodiments of the present invention is notlimited to nucleic acid amplification, but enables loading of a largenumber of solutions onto a microfluidic chip for performance of avariety of applications.

Thus, in certain structures for applying pressure to a elastomericmicrofluidic device comprise, a holder portion including a continuousraised rim on a lower surface thereof configured to contact a topsurface of the microfluidic device and surround a plurality of materialwells located therein. Contact between the raised rim and the topsurface of the microfluidic device defines an airtight chamber over thematerial wells, an orifice in communication with the airtight chamberenabling application of positive pressure to the airtight chamber todrive the contents of the material wells into an active area of themicrofluidic device.

Utilizing such holders, certain methods for priming a microfluidicdevice with a liquid material comprises loading a plurality of wells onan upper surface of a microfluidic device with a liquid material. Aholder piece is biased against the upper surface such that a continuousraised rim of the holder piece presses against the upper surfacesurrounding the wells, such that an airtight chamber is created over thewells. A positive pressure is applied to the airtight chamber to drivethe material from the wells into an active area of the elastomericmicrofluidic structure.

An embodiment of a method of actuating a valve within a microfluidicelastomer device comprises applying a holder piece having a continuousraised rim against a surface of a microfluidic device having a pluralityof control line outlets to create an airtight chamber over the outlets.A positive or negative pressure is applied to the airtight chamber tocontrol a pressure within the control line and thereby actuate aelastomeric valve membrane of the microfluidic device that is incommunication with the control line.

XI. Exemplary Applications

A. General

In some instances, the microfluidic devices described herein can be usedas an analytical tool to amplify a target nucleic acid potentiallypresent in a sample and then detect the amplified product to determinewhether the target nucleic acid is present or absent in the sample.Thus, amplification serves to enhance the ability to detect targetnucleic acids present at low levels. When utilized in this manner, thedevices can be used in a wide variety of different applications. Forexample, the devices can be used in various diagnostic applications thatinvolve a determination of whether a particular nucleic acid is presentin a sample. Hence, samples can be tested for the presence of aparticular nucleic acid associated with particular pathogens (e.g.,certain viruses, bacteria or fungi), for instance. The devices can alsobe utilized for identification purposes, such as in paternity andforensic cases.

The methods and devices provided herein can also be utilized to amplifylow levels of nucleic acid for further examination to detect orcharacterize specific nucleic acids that are correlated with infectiousdiseases, genetic disorders or cellular disorders (e.g., oncogenesassociated with cancer). Genetic disorders are those that involve aspecific deletion and/or mutation in genomic DNA. Examples of geneticdiseases that can be detected include, but are not limited to, α- andβ-thalassemia, cystic fibrosis and sickle cell anemia. Because thedevices and methods disclosed herein can utilize very small samplevolumes, they are useful in amplifying DNA samples obtained inconjunction with the prenatal diagnosis of genetic disease.

However, the amplification reactions can also be utilized as just onestep of a more extensive process involving the diagnostic testing forparticular target nucleic acids and in preparing sufficient nucleic acidfor use in various genetic engineering applications. Hence, amplifiedsample can be used in a number of post amplification manipulations andanalyses. Examples of such post amplification processes and analysesinclude, but are not limited to, sequencing of amplified products,cell-typing, DNA fingerprinting and mapping of DNA sequences.

Amplified products can also be generated for use in various geneticengineering applications. For instance, amplified product can beutilized to conduct recombination studies. In other applications, thedevices are used to produce target DNA for insertion into a variety ofvectors. Such vectors can then be used to transform cells for theproduction of desired products such as proteins, or nucleic acids invarious therapeutic or biotechnological processes.

B. Sequencing

The devices can also be utilized to conduct sequencing reactions such aschain termination methods using dideoxynucleotides. Sequencing reactionsutilizing the devices disclosed herein can be conducted in differentformats. One approach is to conduct four separate sequencing reactions,a separate reaction being conducted in four different thermal cyclingdevices as provided herein. Each of the four reactions contains targetnucleic acid, a primer complementary to the target, a mixture of onedideoxynucleotide (ddNTP) (optionally labeled) with its counterpartdeoxynucleotide (dNTP), and the other three dNTPs. Thus, each one of thereactions is conducted with a different ddNTP/dNTP mix. Followingcompletion of the primer extension reactions, the different sizedextension products can be separated by capillary gel electrophoresis.This separation can be performed in a separation module as describedsupra that is integrated with the present devices or in a stand alonecapillary gel electrophoresis apparatus.

The devices can also be utilized in more streamlined formats in whichreactions are conducted simultaneously in a single thermal cyclingdevice using differentially labeled dideoxynucleotides. The resultingmixture of chain-terminated reaction products are then separated on asingle capillary gel electrophoresis column. The identity of thedideoxynucleotide incorporated into the primer can be determined on thebasis of the label.

C. Restriction Digests

Restriction digests of nucleic acids can also be conducted with thepresent devices. Temperature control in such reactions initiallyinvolves controlling the temperature within the device at a temperaturethat promotes the activity of the restriction enzyme (e.g., 1-3 hours at30-50° C. depending upon the particular enzyme. The other temperature isselected to promote enzyme deactivation (e.g., 60° C. for 20 minutes).

D. SNP Analyses

Analyses to determine the identity of a nucleotide present at apolymorphic site (i.e., the site of variation between allelic sequences)can also be conducted with certain of the present devices. Often theseanalyses are conducted using single base pair extension (SBPE) reaction.A number of SPBE assays have been developed, but the general approach isquite similar. Typically, these assays involve hybridizing a primer thatis complementary to a target nucleic acid such that the 3′ end of theprimer is immediately 5′ of the variant site or is adjacent thereto.Extension is conducted in the presence of one or more labelednon-extendible nucleotides that are complementary to the nucleotide(s)that occupy the variant site and a polymerase. The non-extendiblenucleotide is a nucleotide analog that prevents further extension by thepolymerase once incorporated into the primer. If the addednon-extendible nucleotide(s) is(are) complementary to the nucleotide atthe variant site, then a labeled non-extendible nucleotide isincorporated onto the 3′ end of the primer to generate a labeledextension product. Hence, extended primers provide an indication ofwhich nucleotide is present at the variant site of a target nucleicacid. Such methods and related methods are discussed, for example, inU.S. Pat. Nos. 5,846,710; 6,004,744; 5,888,819; 5,856,092; and5,710,028; and in WO 92/16657.

Using devices as described herein, the temperature within a temperaturecontrol region can be selected to promote the primer annealing, primerextension and denaturation steps involved in these particular analyses,and thus allows these extension reactions to be conducted in a thermalcycling format.

E. Non-Nucleic Acid Analyses

While the foregoing discussion of the matrix or array microfluidicdevices has focused on their utility in conducting a large number ofnucleic acid amplification reactions, it will be appreciated by thosewith ordinary skill in the art that such microfluidic devices can beutilized to conduct a wide variety of types of reactions and screeningmethods. Thus, by way of illustration but not limitation, the devicescan be utilized to conduct synthetic reactions between a plurality ofreactants. Using the device shown in FIG. 12, for instance, a first setof reagents can be introduced into the horizontal flow channels, asecond set of reagents can be introduced into the vertical flow channelsthat have an independent inlet; and a third reagent can be introducedinto the vertical flow channels that are connected to the shared inlet.Using the metering technique discussed above, these various reagents canbe combined within the junctions or reactant chambers for reaction.

The devices can also be utilized to screen compounds for a desiredactivity. With the devices described in FIGS. 5 and 12 for example,typical screening methods involve introducing a set of test compoundsinto the horizontal flow channels, with another set of compounds, cells,vesicles or the like being introduced via the vertical flow channels.

Mixing occurs at the junctions and the presence or absence of thedesired activity can then be monitored at the junction.

For instance, a wide variety of binding assays can be conductedutilizing the microfluidic devices disclosed herein. Interactionsbetween essentially any ligand and antiligand can be detected. Examplesof ligand/antiligand binding interactions that can be investigatedinclude, but are not limited to, enzyme/ligand interactions (e.g.,substrates, cofactors, inhibitors); receptor/ligand; antigen/antibody;protein/protein (homophilic/heterophilic interactions); protein/nucleicacid; DNA/DNA; and DNA/RNA. Thus, the assays can be used to identifyagonists and antagonists to receptors of interest, to identify ligandsable to bind receptors and trigger an intracellular signal cascade, andto identify complementary nucleic acids, for example. Assays can beconducted in direct binding formats in which a ligand and putativeantiligand are contacted with one another or in competitive bindingformats well known to those of ordinary skill in the art. Binding assayscan be conducted in heterogenous formats, as well as homogenous formats.In the homogeneous formats, ligands and antiligands are contacted withone another in solution and binding complexes detected without having toremove uncomplexed ligands and antiligands. Two approaches frequentlyutilized to conduct homogenous assays are fluorescence polarization (FP)and FRET assays.

Immunological assays are one general category of assays that can beperformed with certain of the microfluidic devices disclosed herein.Some assays are conducted to screen a population of antibodies for thosethat can specifically bind to a particular antigen of interest. In suchassays, a test antibody or population of antibodies is contacted withthe antigen. Typically, the antigen is attached to a solid support.Examples of immunological assays include enzyme linked immunosorbentassays (ELISA) and competitive assays as are known in the art.

A variety of enzymatic assays can be performed using some of the devicesdisclosed herein. Such enzymatic assays generally involve introducing anassay mixture containing the necessary components to conduct an assayinto a flow channel or jucntion for reaction with an enzyme that issubsequently introduced. The assay mixtures typically contain thesubstrate(s) for the enzyme, necessary cofactors (e.g., metal ions,NADH, NAPDH), and buffer, for example. If a coupled assay is to beperformed, the assay solution will also generally contain the enzyme,substrate(s) and cofactors necessary for the enzymatic couple.

A number of different cell reporter assays can be conducted with theprovided microfluidic devices. One common type of reporter assay thatcan be conducted include those designed to identify agents that can bindto a cellular receptor and trigger the activation of an intracellularsignal or signal cascade that activates transcription of a reporterconstruct. Such assays are useful for identifying compounds that canactivate expression of a gene of interest. Two-hybrid assays, discussedbelow, are another major group of cell reporter assays that can beperformed with the devices. The two-hybrid assays are useful forinvestigating binding interactions between proteins.

Cells utilized in screening compounds to identify those able to triggergene expression typically express a receptor of interest and harbor aheterologous reporter construct. The receptor is one which activatestranscription of a gene upon binding of a ligand to the receptor. Thereporter construct is usually a vector that includes a transcriptionalcontrol element and a reporter gene operably linked thereto. Thetranscriptional control element is a genetic element that is responsiveto an intracellular signal (e.g., a transcription factor) generated uponbinding of a ligand to the receptor under investigation. The reportergene encodes a detectable transcriptional or translational product.Often the reporter (e.g., an enzyme) can generate an optical signal thatcan be detected by a detector associated with a microfluidic device.

In addition to the assays just described, a variety of methods to assayfor cell membrane potential can be conducted with the microfluidicdevices disclosed herein. In general, methods for monitoring membranepotential and ion channel activity can be measured using two alternatemethods. One general approach is to use fluorescent ion shelters tomeasure bulk changes in ion concentrations inside cells. The secondgeneral approach is to use of FRET dyes sensitive to membrane potential.

Assays of cell proliferation can also be monitored with certain of themicrofluidic devices disclosed herein. Such assays can be utilized in avariety of different studies. For example, the cell proliferation assayscan be utilized in toxicological analyses, for example. Cellproliferation assays also have value in screening compounds for thetreatment of various cell proliferation disorders including tumors.

The microfluidic devices disclosed herein can be utilized to perform avariety of different assays designed to identify toxic conditions,screen agents for potential toxicity, investigate cellular responses totoxic insults and assay for cell death. A variety of differentparameters can be monitored to assess toxicity. Examples of suchparameters include, but are not limited to, cell proliferation,monitoring activation of cellular pathways for toxicological responsesby gene or protein expression analysis, DNA fragmentation; changes inthe composition of cellular membranes, membrane permeability, activationof components of death-receptors or downstream signaling pathways (e.g.,caspases), generic stress responses, NF-kappaB activation and responsesto mitogens. Related assays are used to assay for apoptosis (aprogrammed process of cell death) and necrosis.

By contacting various microbial cells with different test compounds,some of the devices provided herein can be used to conduct antimicrobialassays, thereby identifying potential antibacterial compounds. The term“microbe” as used herein refers to any microscopic and/or unicellularfungus, any bacteria or any protozoan. Some antimicrobial assays involveretaining a cell in a cell cage and contacting it with at least onepotential antimicrobial compound. The effect of the compound can bedetected as any detectable change in the health and/or metabolism of thecell. Examples of such changes, include but are not limited to,alteration in growth, cell proliferation, cell differentiation, geneexpression, cell division and the like.

Additional discussion of biological assays that can be conducted withcertain of the microfluidic devices disclosed herein is provided incommonly owned PCT application PCT/US01/44869, filed Nov. 16, 2001.

The following examples are provided to describe in greater detailcertain aspects of the methods and devices disclosed herein.

Example 1

This example illustrates one method of fabricating a microfluidic devicecomprising a rotary microfluidic channel and a temperature controller(e.g., heater).

A glass slide was gas phase treated with HMDS and Shipley SJR 574 wasspun onto the glass slide. After photolithography and developing,tungsten was sputtered on the slide after removal of the photoresist(under the tungsten) with acetone. Contacts between the electrical linesrunning from the power supply and the heaters are made with conductiveepoxy (Chemtronics, Kennesaw, Ga.).

A device having the configuration shown in FIG. 1 was prepared asfollows. Air and fluid mother molds (for formation of control and flowchannels, respectively) were fabricated on silicon wafers byphotolithography. Photoresist (Shipley SJR5740) was spun onto thesilicon substrate at spin rates corresponding to the desired channelheights. After photolithography, intrusive channels made of photoresistwere formed. Fluid channel molds were baked on a hot plate of 200° C.for 30 minutes such that the photoresist could reflow and form a roundedshape, which is important for a complete valve closure (see, M. A.Unger, et al. (2000) Science 288:113-116). A one minutetrimethylchlorosilane (TMCS) vapor treatment was applied to these moldsbefore each RTV replication process to prevent adhesion of cured RTV tothe photoresist. With this protective coating, molds can be reused manytimes.

30:1 GE-RTV 615A:615B was spun on a fluid channel mold at 2,000 RPM,which covers the photoresist channel and leave a thin membrane on top ofit. At the same time, 3:1 GE-RTV 615A:615B was poured onto an airchannel mold. After baking both in an oven of 80° C. for 20 minutes, theblock of 3:1 RTV with air channels at the bottom was peeled off from thesecond mold. Air supply through-holes were punched. Aligned to the fluidpattern under a microscope, it was then pressed against the thin 30:1RTV on the first mold. A post-bake of an hour at 80° C. made the twosilicone pieces chemically bond to each other. After peeling it off fromthe mold and punching the fluid through-holes, the monolithic RTV devicewas subjected to an HCl treatment (0.1 N, 30 min. at 80° C.). Thistreatment breaks some Si—O—Si bonds and displays them on the surface ofthe channel. This helps make the polymer more hydrophilic, therebyenhancing solution flow through the channel. The monolithic device wasthen hermetically sealed to the glass cover slip upon which the heatershad previously been sputtered.

As illustrated in FIG. 1, samples containing or potentially containingtarget nucleic acids (e.g., DNA), amplification reagents and agents fordetection of unreacted reagents and/or products can enter from the twobranches of the top T-channel. On/off states of each microvalves can becontrolled by external pneumatic valves (Lee LHDA1211111H) which eitherapply 100-kPa air pressure to the microvalves or vent them to theatmosphere. A maximum cycling frequency of 75 Hz has been demonstratedwith complete opening and closing of the valves (see, e.g., L. C.Waters, et al. (1998) Analytical Chemistry., 70:158).

Example 2

This method illustrate another method of fabricating a microfluidicdevice comprising a rotary microfluidic channel and a temperaturecontroller (e.g., heater).

The rotary microfluidic devices were fabricated by multilayer softlithography techniques (Unger, M. A. et al., Science, 288:113-116(2000); Chou, H. P. et al., Proceedings of the Solid State Sensor andActuator Workshop, Hilton Head, S.C. (2000)). The channel pattern of thedevice is shown in FIG. 7A. As can be seen, the device 700 includes acircular flow channel 702 in fluid communication with an inlet 704 andan outlet 706. The flow channel 702 overlays three resistive heaters 708a, 708 b and 708 c, each defining a different temperature region andhaving a different length. S-shaped control channels 710 a and 710 boverlay the circular flow channel 702 to form pumps for flowing fluidthrough the circular flow channel 702. The control channels 712 a and712 b overlaying the inlet 704 and outlet 706 serve to introduce andwithdraw solution from the main flow channel.

The masks for making the flow channel were printed on transparency filmsby a high resolution printer (3556 dpi). Photoresist (Shipley SJR5740)was spun onto the silicon wafer, which had been pretreated with HDMS, at3000 RPM for 60 seconds. After preheating at 90° C. for 1 hour, thechannel pattern was exposed onto the wafer with a mask aligner (KarlSuss). The pattern was developed by 5:1 (v/v) developing solution(Shipley 2401). Then the wafer was put onto a hot plate for 30 min toround the remaining photoresist.

As represented in FIG. 7B, 20:1 RTV 615(GE) was spun on a flow channelmold at 2,000 RPM; while 5:1 RTV (GE) was spun on a control channelmold, then 10:1 RTV (GE) was poured onto it. After baking them in anoven of 80° C. for 1.5 hour, the RTV block on the latter mold was peeledoff to punch air supply vias. Then it was aligned and pressed againstthe thin RTV layer on the former mold. After a post-bake at 80° C. for 2hours, the multilayer RTV was peeled off the mold to punch fluid vias.Finally, the device was made by sealing the multilayer RTV onto a glasscoverslip at 80° C. for 2 hours.

The on-chip valves were actuated with a pneumatic controller supplied byFluidigm, Inc. The Fluid Controller system allowed selective actuationof valves to seal off the loop as well as peristaltic pumping atvariable rates within the loop. Typical actuation pressures were 10 psi;the pumping frequency was 30 Hz.

The pattern of heaters is shown in FIG. 7B. They were made of thinlayers of tungsten (heating component) and aluminum (electrical leads)sputtered onto a glass microscope slide. Similar to the fluidicfabrication, a negative pattern of photoresist was developed onto apiece of a glass microscope slide (40 mm×26 mm×1.1 mm). Then a thinlayer (˜500 Angstroms) of tungsten was deposited on the sample by a DCsputter system. After that, acetone was used to clean off the remainingphotoresist in order to get the designed tungsten pattern. The procedurewas repeated in order to sputter another thin layer of aluminum as theelectrical leads for the heater.

The heaters were calibrated via a two step process. After the fluidicdevice was attached to the heaters, thermochromic liquid crystals (TLC)were pumped into the channels of the device. The TLC were then imaged atvarious heater currents with a color camera (KR222, Panasonic) and astereomicroscope (ASA012-3449, Japan). The temperature response of theTLC were separately calibrated with a thermistor and hot plate. Briefly,images of the beads were digitized and average hues of the beads werecalculated, with outliers discarded. Hue and temperature have a fairlylinear relationship from 75-100 degrees, sufficient to determine thedenaturing temperature. The lower annealing/extension temperature wasextrapolated from this curve.

During Taqman PCR, the denaturing temperature was set at 95° C., and theannealing temperature at 60° C. Two independent resistive heaters wereused to heat the microchip to set temperatures. The output power of theheaters was adjusted by the voltage: around 380 mW (12.6V) and 75 mW(7.0V), respectively.

Example 3

This example illustrates Taqman PCR assay using the microfluidic deviceof Example 2 having two distinct temperature zones.

The Taqman PCR technique exploits the 5′-3′ nuclease activity ofAmpliTaq Gold DNA polymerase to allow direct detection of PCR product bythe release of a fluorescent reporter during PCR (Protocol of TaqMan PCRReagent Kit with AmpliTaq Gold DNA Polymerase, Applied Biosystems.http://www.appliedbiosystems.com. (2001)). An advantage of using thiskit is that the increase in fluorescence during PCR quantitativelyreflects the amount of product created. The probe was an oligonucleotidecontaining a report dye, 6-carboxyfluorescein (FAM) in the 5′ end and aquencher dye, 6-carboxytetramethylrhodamine, in the 3′ end. Theexcitation wavelength was 488 nm, while the emission wavelength of thereporter dye and quencher dye were 518 nm and 582 nm, respectively. Thetemplate, Human DNA Male, was provided with the kit. The target wasportion of the β-actin gene; forward primer and reverse primer were thefollowing: 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′ and 5-′CAG CGG AACCGC TCA TTG CCA ATG G-3′, respectively. A segment of DNA about 294 bpwas amplified. Typical PCR reagent conditions were as follows (50-μLvolumes): template, 0.1 ng/μL; primers, 300 nM; Mg²⁺, 3.5 mM; dATP, 200μM; dCTP, 200 μM; dGTP, 200 μM; dUTP, 400 μM; AmpliTaq Gold, 0.025 U/μL;AmpErase UNG, 0.01 U/μL; Probe, 200 nM. A benchtop PCR machine (PTC200Peltier Thermal Cycler, MJ Research) was used to check the Taqman PCRkit by the manufacturer's protocol: 2 minutes at 50° C.; 10 minutes at95° C.; 40 cycles: 15 seconds at 95° C.; 1 minute at 60° C.

FIGS. 8 and 9A show the emission spectrum and gel electrophoresisresults, respectively, of the PCR product by ordinary PCR machine. Theemission spectra in FIG. 8 show the sample and the negative control (notemplate). The fluorescent peak at 525 nm appears in the sample with thetemplate, while there is no peak for the control. In FIG. 9A, lane 1(left) is that of human β-actin Taqman PCR amplicon: 123 bp DNA ladder;lane 3 (middle) is that of a sample; and lane 5 (right), which is nearlyinvisible is no template control.

Example 4

This example illustrates Taqman PCR assay using the microfluidic deviceof Example 2 having three temperature zones.

A segment (199-bp) of λ phage DNA was selected for amplification;forward primer and reverse primer were the following: 5′-GGT TAT CGA AATCAG CCA CAG CGC C-3′ and 5′-GGA TAC GTC TGA ACT GGT CAC-3′, respectively(Khandurina, J. et al., Anal. Chem., 72:2995-3000 (2000)). Typical PCRreagent conditions were as follows (100-μL volumes): template, 2 ng/μL;primers, 500 nM; Mg²⁺, 1.5 mM; dNTP, 200 nM; Taq polymerase, 0.025 U/μL(Qiagen); An intercalating dye, Sybr Green I (Molecular Probes), wasadded to the reagent mixture using a 1:10,000 dilution of the stockreagent.

For amplification by benchtop PCR machine, the temperature steps wereset as follows: 2 minutes at 94° C.; 30 cycles: 30 seconds at 94° C.; 30seconds at 55° C.; 1 minute at 72° C.; and 10 minutes at 72° C. finally.FIG. 9B shows the gel electrophoresis result of the PCR product of λphage DNA by this method. In FIG. 9B, lane 1 (left) is that of 123 bpDNA ladder; lane 2 (middle) is that of λ-DNA amplicon; and lane 3(right) is that of no template control. The sample and the control weredisplayed in 2% Agarose gel (Model 1A, Easy-cast™). All samples weretreated with Sybr Green I stain. The picture was taken by the Kodakelectrophoresis documentation and analysis system.

Example 5

This example illustrates real-time measurement of PCR products using amicrofluidic device of Example 2.

After having injected the reagent mixture into the microchip, the rotarymicrofluidic channel (i.e., loop) circulation was operated with theFluid Controller (Fluidigm) and turned on the power supply for theheaters. The fluorescence change inside the channel was imaged by a highresolution camera (ST-7, Santa Babara Instruments Group) assembled on afluorescence microscope (IX50, Olympus) every a few minutes during theprocess of PCR. The filters (Chroma) in the fluorescence microscope werechosen as follows: Exciter, HQ 470/40x; Dichroic, Q 495LP; Emitter, HQ525/50m. The snap pictures were analyzed with the CCDOPS (SBIG)software.

As shown schematically in FIG. 7, the flow channel is a central loop(i.e., rotary microfluidic channel) with an inlet and an outlet. In thisparticular example, the channel width of the central loop isasymmetrical: 120 mm on the left; 70 μm on the right. In such a pattern,the fluid velocities in the two parts are different from each other.Therefore, the time that the fluid resides in different temperaturezones can be adjusted either by the width of flow channel or the lengthof the heater. The fluid motion in the channel was characterized byusing a solution of 2.5 μm beads. In order to mimic the actualconditions of the assay, we used the same concentration of Taqman PCRbuffer and Human DNA (male) in the bead solution. At room temperature,the speed of beads was about 3.4 mm/second in the narrow channel and 1.5mm/second in the wide channel. However, it was slowed down to 25%-30% ofthe above velocity when the device was run at its operatingtemperatures, resulting in about 20˜30 seconds per full cycle.

In the control layer, three separate valves were used to create theperistaltic pump. Alternatively, one S-shaped valve can also accomplishthe same peristaltic pumping as three separate valves. In addition, theS-shape channels provide more secure closing of the inlet and outletthan a single channel.

In each of the experiments one portion of the flow channel was selectedto observe the fluorescence change. The CCDOPS software provided a wayto get the intensity values inside the channel and outside the channelby sampling the different regions of the picture. The intensity valueoutside the channel was subtracted as a measure of the background.

Spatial Cycling

The microfluidic device described above was used in the spatial cyclingmode to amplify a fragment of the human β-actin gene using the Taqmanassay. After running the rotary pump in the microchip for about 10minutes, a rapid increase in fluorescence was observed. As shown in FIG.10, there was a more than four fold increase in fluorescence intensityin the presence of the sample template, while the no-template controlremained flat. In FIG. 10, dot represents sample and square representsno template control. The curves are a guide for the eye.

Calibration experiments showed that the recorded increase influorescence was comparable to that of 30˜35 cycles of PCR product by abenchtop PCR machine (PTC200 Peltier Thermal Cycler, MJ Research) (FIG.10).

The microfluidic device was also used to perform a three temperature PCRassay in which the creation of product was directly monitored byobserving the product via an intercalating dye. The reagents were pumpedthrough three different temperature zones as follows: denaturing (94°C.) and annealing (55° C.) extension (72° C.). A fragment of λ DNA wasamplified by Taq Polymerase while the PCR product was investigated withSybr Green I. FIG. 11A shows the fluorescence difference before andafter PCR process, and indicates that some amplification occurs relativeto the no template control. FIG. 11A shows fluorescence measured in thechannel before and after 40 minutes of pumping, where the dark barrepresents PCR process with DNA template and the white bar representscontrol without template.

Temporal Cycling

The same segment of λ phage DNA was amplified using the microfluidicdescribed above. After having injected the reagent mixture into themicrofluidic device, the inlet and outlet valves were closed. Thecentral loop was heated and cooled repeatedly without circulating thesample within the loop. The heaters were calibrated to the settemperatures by choosing a set of voltages; the temperature steps wereas follows: 15 seconds at 94° C.; 30 seconds at 55° C.; and 30 secondsat 72° C. Each cycle took about 6 minutes and 23 cycles were conductedin the experiment. The change of fluorescence during PCR was monitoredby the same setup every a few cycles. The fluorescence increaseddramatically during the experiment as shown in FIG. 11B.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

1. A microfluidic device, comprising: (a) a substrate comprising anelastomeric material; (b) a flow channel disposed within the substrate,the flow channel configured such that a sample introduced, into the flowchannel can be cycled around the flow channel; and comprising aplurality of temperature regions at which temperature can be regulated,each temperature region located at a different location along the flowchannel; (c) an inlet in fluid communication with the flow channel viawhich the sample can be introduced into the flow channel; and (d) atemperature controller operatively disposed to regulate temperaturewithin at least one of the plurality of temperature regions. 2-99.(canceled)